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Introduction
Cysteinyl leukotrienes (CysLT; including
LTC4, LTD4 and
LTE4), 5-lipoxygenase (5-LOX) metabolites of arachidonic acid,
are the potent inflammatory mediators involved in various central nervous diseases, such as cerebral
ischemia[1_3], brain
trauma[4], brain tumors[5] and
epilepsy[6]. After cerebral ischemia or ischemia-reperfusion, the production of CysLT in the
ischemic brain is increased[1_3]. In addition, 5-LOX inhibitors ameliorate cerebral ischemic injuries via reducing the
production of CysLT[7,8]. Since the actions of CysLT are mediated by activating their receptors (CysLT receptors), it is important to
investigate the changes in CysLT receptors after cerebral ischemia.
Two types of CysLT receptors,
CysLT1 and CysLT2, have been cloned and identified as G protein-coupled
receptors[9_14]. The CysLT1 receptor is highly expressed in the human spleen and peripheral blood leukocytes, and weakly in the brain, lung,
colon, heart, pancreas, prostate, skeletal muscle, kidney, liver, placenta and small intestine as determined by Northern
blotting[9,10]. The mouse
CysLT1 receptor has an expression pattern distinct from that of humans; it is highly expressed
in the lung and skin, and weakly in other tissues including the
brain[15]. Little is known about the expression and localization of the
CysLT1 receptor in the brain. Recently, we reported that the
CysLT1 receptor was primarily expressed in the microvascular
endothelia in the human brain by immunohistochemical analyses, and its expression was increased in the neuron- and
glial-appearing cells after traumatic
injury[16]. Moreover, we have found that the
CysLT1 receptor antagonists (pranlukast and
montelukast) protect against cerebral ischemic injury in rats and mice, pharmacologically suggesting that the
CysLT1 receptor may play a role in cerebral
ischemia[17_22].
On the other hand, the
CysLT2 receptor is highly expressed in the spleen, heart, adrenal glands and thymus, and weakly
in the kidney, brain and peripheral blood
leukocytes[23]. In the brain, the
CysLT2 receptor expression is found in most regions
and in the spinal cord[12,13]. The expression patterns of the
CysLT2 receptor are similar in mouse and human tissues. We found
that the CysLT2 receptor was primarily expressed in the smooth muscle cells of cerebral vessels in the human brain by
immunohistochemical analyses, and its expression was also increased in the neuron- and glial-appearing cells after traumatic
injury[24]. Because no selective antagonist is available for the
CysLT2 receptor, its role in the brain is unknown.
However, no direct evidence shows the role of
CysLT1 and CysLT2 receptors in the brain after cerebral ischemia. To clarify
their role in acute ischemic neuronal injury, we investigated the temporal expressions of
CysLT1 and CysLT2 receptor mRNA
in the mouse brain from 1 h to 48 h (acute phase) after focal cerebral ischemia. We wanted to show the relationship between
the temporal expressions and the development of ischemic neuronal injury. In addition, we observed the localization of the
CysLT1 receptor 24 h after ischemia.
Materials and methods
Physiological parameter
monitoring Male ICR (Institute of Cancer Researcch) mice weighing 25_30 g (Shanghai
Experimental Animal Center, China, Shanghai, China; Certificate
No. 22-001004) were used. The mice were housed under a
controlled temperature, 12 h light/12 h dark cycle and allowed access to food and water
ad libitum. All experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.
The mice were anaesthetized with chloral hydrate (400 mg/kg, ip). A polyethylene tube was inserted into the right femoral
artery for continuous monitoring of blood pressure using a computer-assisted system (PC-Lab, Kelong Inc, Nanjing, China),
and for measuring arterial blood
Pao2, Paco2 and pH (Blood Gas Analyzer ABL 330, Leidu Inc, Copenhagen, Kobenhavn,
Denmark). Blood glucose was monitored by a one touch basic blood glucose monitoring system (Lifescan Inc, New Brunswick,
New Jersey, 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.
Permanent focal cerebral ischemia
After anesthesia, permanent focal cerebral ischemia was induced by middle
cerebral artery occlusion (MCAO) as described
previously[17]. 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 the sham-operated mice, the same procedure was
done, except for the insertion of the intraluminal filament. After surgery, the mice were kept for about
2 h in a warm box heated by lamps to maintain the body temperature.
Behavioral assessments Neurological deficit scores were evaluated 6, 24 and 48 h after
MCAO[25]: 0, no deficit; 1, flexion of contralateral forelimb upon lifting of the whole animal by the tail; 2, circling to the contralateral side; 3, falling to
contralateral side; and 4, no spontaneous motor activity. An inclined board test was performed to assess balance and
coordination[20] based on modification of a reported
method[26]. The mice were placed on a board (50×30 cm); once they were stable, the board
was inclined from horizontal to vertical. 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. Behavioral assessments were not observed 1 and 3 h after
ischemia because the mice did not recover completely from the anesthesia.
Histopathology analyses The brains were removed at 1, 3, 6, 24, and 48 h after MCAO, fixed in 4% paraformaldehyde
overnight, and cut by cryostat into serial 8 mm-thick coronal sections, then stained with 1% toluidine blue. The apparently
surviving neuron-appearing cells in the temporoparietal cortex III, IV layers, the hippocampal CA1 region and the striatum
(1.8_2.0 mm caudal from the bregma) were counted using the ImageTool 2.0 analysis program (University of Texas Health
Science Center in San Antonio, San Antonio, Texas, USA).
RT-PCR The ischemic and the contralateral non-ischemic hemispheres were dissected on ice 1, 3, 6, 24 and 48 h after
MCAO, and stored at -80 °C for RNA extraction. Total RNA was extracted using Trizol reagents (Invitrogen, Carlsbad,
California,USA) according to the manufacture's protocol. For cDNA synthesis, aliquots of total RNA (1 µg) were mixed with
1 mmol/L dNTP, 0.2 µg random primer, 20 U RNase, 200 U M-MuLV reverse transcriptase in 20 µL of the reverse reaction
buffer. The mixture was incubated at 42 °C for 60 min, and then 70 °C for 10 min to inactivate the reverse trans-criptase. As
a negative control, the reaction was performed at the absence of reverse transcriptase.
PCR was performed in a 40 µL reaction mixture containing PCR buffer, 4 µL cDNA, 200 µmol/L dNTP, 1.5 mmol/L
MgCl2, 10 pmol of each primer and 1 U of Taq DNA polymerase (Takara, Kyoto, Japan). PCR reactions were performed as follows: 94
ºC for 2 min followed by 35 cycles of 93 ºC for 10 s, 66 ºC for 30 s and 72 ºC for 30 s, and finally extended at 72 ºC for 10 min. The
primer sequences for mouse CysLT1 and
CysLT2 receptors[13] were synthesized by Sangon Biotech Co (Shanghai, China) as
follows: mCysLT1 (+): 5'-CAA CGA ACT ATC CAC CTT CAC C-3',
mCysLT1 (_): 5'-AGC CTT CTC CTA AAG TTT CCA C-3';
mCysLT2 (+): 5'-GTC CAC GTG CTG CTC ATA GG-3',
mCysLT2 (_): 5'-ATT GGC TGC AGC CAT GGT C-3'. GAPDH was used
as the control; its primer sequences were GAPDH (+): 5'-GTC GGT GTG AAC GGA TTT GG-3' and GAPDH (_): 5'-GCT CCT
GGA AGA TGG TGA TGG-3'. PCR products in 10 µL were separated by electrophoresis on a 2% agarose gel containing
ethidium bromide and photographed. The optical density of the bands was determined by an image analysis system
(Bio-Rad, Hercules, California, USA). The PCR product sizes of
CysLT1, CysLT2 receptors and GAPDH were162 bp, 180 bp and 223
bp, respectively (Figure 1A). CysLT1 and
CysLT2 receptor mRNA levels were normalized to the level of GAPDH mRNA.
Double
immunofluorescence To visualize the localization of the
CysLT1 receptor, double immunofluorescence was
employed. Briefly, after blocking non-specific binding of IgG with 5% normal goat serum for 2 h at room temperature, the brain
sections were incubated overnight at 4 °C with a mixture of rabbit polyclonal anti-human
CysLT1 receptor antibody (1:150, Cayman Chemicals, Ann Arbor, MI, USA) and mouse monoclonal antibody against neuronal nuclei (NeuN; 1:200; Chemicon,
Temecula, California, USA), a specific marker of neurons, or against a glial fibrillary acidic protein (GFAP; 1:800; Chemicon,
USA), a specific marker of astrocytes. Then, the sections were incubated with the mixture of fluorescein isothiocyanate
(FITC)-conjugated goat anti-rabbit IgG and Cy3-conjugated goat anti-mouse IgG
(1:100; Chemicon, USA).
To confirm the specificity of the polyclonal rabbit anti-human
CysLT1 receptor antibody used in the double
immunofluorescence of the mouse brain tissue, we collected protein samples of cultured human neuroblastma cell line SK-N-SH cells
(purchased from the Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, China) as well as a normal mouse lung
and cerebral cortex. Protein samples (50 mg) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred
to nitrocellulose membranes. The membranes then reacted with the rabbit polyclonal anti-human
CysLT1 receptor antibody (1:2000), and with the peroxidase-conjugated goat anti-rabbit IgG (1:2000) after repeated washing. Finally the protein bands
were visualized by enhanced chemiluminescence. The result showed that a band was close to 43 kDa in the mouse cerebral
cortex, the same as that in the SK-N-SH cells (Figure 1B). This was consistent with previously published results using the
same polyclonal antibody[27,28]. Thus, the antibody was usable for immunohistochemistry of mouse brain tissue.
Statistical analysis Data are presented as mean±SD. Differences between groups were analyzed by one-way ANOVA
(Student-Newman-Keuls) using the SPSS softwarepackage (Version 10.0 for Windows; SPSS, Chicago, Illinois, USA).
P<0.05 was considered statistically significant.
Results
Physiological variables
There was no significant difference in mean arterial blood pressure,
PaO2, PaCO2, arterial blood pH,
and blood glucose between 30 min before and 30 min after ischemia (Table 1).
Ischemic injury The neurological deficit score was increased (Figure 2A) and the holding angle in the inclined board test
was reduced (Figure 2B) 6, 24, and 48 h after MCAO. In the cortex, the hippocampal CA1 region and the striatum of the
ischemic hemisphere, no apparent change was observed, and the number of neuron-appearing cells was unchanged at 1 and
3 h after MCAO. The Nissl bodies in the neurons disappeared and the neurons shrunk at 6 h after MCAO; most of neurons
were destroyed and left vacuoles at 24 and 48 h after MCAO (Figure 3A). The number of neuron-appearing cells in these
regions was significantly reduced at 6, 24, and 48 h after MCAO (Figure 3B). As the control, the cell number in the
contralateral non-ischemic hemispheres had no significant change within 48 h after MCAO.
Temporal expressions of
CysLT1 and CysLT2 receptor mRNA
The expression of CysLT1 receptor mRNA in the ischemic
hemisphere initially increased at 1 (1.9-fold) and 3 h (1.6-fold), partially recovered at 6 h, and largely increased again 24
(4.5-fold) and 48 h (3.8-fold) after MCAO (Figure 4A,C). Similarly, the expression of
CysLT2 receptor mRNA in the ischemic hemisphere significantly increased at 1 h (2.2-fold), partially recovered at 3_6 h, then largely increased again 24 (5.3-fold) and
48 h (3-fold) after MCAO (Figure 4B,4D).
CysLT1 receptor
localization Double immunofluorescence staining showed that the
CysLT1 receptor was localized in the
NeuN-positive neurons in the cortex, the hippocampal CA1 region and the striatum of the ischemic hemispheres at 24 h
after MCAO (right panels in Figure 5A); almost none were detected in the hemisphere from the
sham-operated mice (left panels in Figure 5A). On the other hand, the
CysLT1 receptor was not localized in GFAP-positive astrocytes in these regions
in both the sham-operated and ischemic hemispheres (Figure 5B).
Discussion
In the present study, the most important finding is that both the expressions of
CysLT1 and CysLT2 receptor mRNA in the
ischemic brain are upregulated in the acute phase of focal cerebral ischemia. The increased expressions are temporally related
to neuronal injury, including neurological deficits and regional neuron loss. These results suggest that both
CysLT1 and CysLT2 receptors may be involved in acute ischemic neuronal injury.
Because neuronal injury occurs initially in the acute phase after cerebral ischemia, in this study, we focus on the temporal
changes in CysLT1 and CysLT2 receptor mRNA as
well as the neuronal injury within 48 h after focal cerebral ischemia. Neurological deficits and neuron loss appeared from 6 to
48 h (Figures 2, 3). Both CysLT1 and
CysLT2 receptor mRNA were initially increased at 1 h, and largely increased at 24 and 48
h after ischemia with peaks at 24 h (Figure 4). We can not explain why both the receptor mRNA expressions temporally
increased, however, the initial increase might be induced by excitotoxicity that occurs at minute to hour after cerebral
ischemia, and might mediate the initiation of delayed
responses (hour to day), such as neuronal injury and
inflammation[29]. As supporting evidence, we found that microinjection of
N-methyl-D-aspartate (an exogenous excitatory acid) into the mouse
cortex induced CysLT1 receptor
expression (unpublished observations). On the other hand, the delayed increase of the 2
receptor mRNA expressions (24 and 48 h after MCAO) might be induced in response to secondary inflammation, and might
mediate late responses like neuronal injury. The possible mediation was suggested by a close relationship between the
increased expressions and the neuronal injury 24 and 48 h after MCAO in the present study. Of course, ischemic neuronal
injury is caused by more complex systems, not by the
CysLT1 or CysLT2 receptor alone.
The novel finding is that the expression of
CysLT2 receptor mRNA is upregulated after focal cerebral ischemia. However,
because neither selective antagonists nor specific antibodies (for rats and mice) are available for studying the
CysLT2 receptor, we can not explore more properties of this receptor in mice or rats with cerebral ischemia. In peripheral tissues, the
CysLT2 receptor mediates endothelial and macrophage activation, mast cell interleukin-8 secretion, and
fibrosis[30], but it is not known how the
CysLT2 receptor regulates the central nervous function. Recently, we showed that the
CysLT2 receptor expression increased in neuron-appearing cells as detected by immunohistochemical staining in human brain specimens from
patients with traumatic brain
injury[24], but the implication is still unclear. We also found that PC12 cells transfected with the
CysLT2 receptor were more sensitive than those transfected with the
CysLT1 receptor to the in vitro ischemic-like injury
induced by oxygen-glucose
deprivation[31], suggesting that the
CysLT2 receptor may play a more important modulating role
in ischemic neuronal injury.
Another important finding is that the
CysLT1 receptor is localized in the neurons in the ischemic brain and may mediate
neuronal injury as confirmed by CysLT1 receptor antagonism. For detecting
CysLT1 receptor localization, we performed
double immunofluorescence using a polyclonal rabbit anti-human
CysLT1 receptor antibody, which was also specific to
mouse brain tissue (Figure 1B). Much less
CysLT1 receptor is expressed in the normal brain from the sham-operated mice, but
it was highly expressed in the neurons in the ischemic cortex, the hippocampal CA1 region and the striatum 24 h after focal
cerebral ischemia. Furthermore, the
CysLT1 receptor was less expressed in astrocytes. A similar pattern of
CysLT1 receptor localization was also found in the rat brain after transient focal cerebral ischemia in our recent study (unpublished observations).
In that study, we found that the CysLT1 receptor was primarily expressed in neurons, not in astrocytes, in the cortex 24 h after
transient focal cerebral ischemia, while it was expressed in the astrocytes, not in neurons, in the boundary zone adjacent to
the ischemic core 14 d after ischemia. The present finding further indicated the localization of the
CysLT1 receptor in the striatum and the hippocampal CA1 region in addition to the cortex in a mouse model of permanent focal cerebral ischemia
without reperfusion. Both the results from the focal ischemic mice
and rats indicate that the CysLT1 receptor may mediate
neuron injury in the acute phase of ischemia. This mediation has been suggested by the neuroprotective effect of selective
CysLT1 receptor antagonists as we previously
reported[17_20], although the antagonists may have other non-specific effects
attenuating ischemic injury. The
CysLT1 receptor regulates vascular leak, bronchoconstriction, dendritic cell maturation and
migration, macrophage activation, hematopoietic progenitor cell recruitment, eosinophil and mast cell cytokine secretion, and
smooth muscle proliferation in peripheral
tissues[30]. Our finding indicates a role of the
CysLT1 receptor in ischemic neuronal injury.
In summary, the present study shows that
CysLT1 and CysLT2 receptors are upregulated in acute neuronal injury after
focal cerebral ischemia; The CysLT1
receptor is localized in neurons after ischemia. Since inflammation is one of the
post-ischemic events in the brain[29] and pro-inflammatory CysLT are produced in the brain after cerebral
ischemia[1_3],
it is necessary to clarify the modulating roles of
CysLT1 and CysLT2 receptors in cerebral ischemia. Our findings of
CysLT1 and CysLT2 receptor expression will contribute to understanding the mechanisms of cerebral ischemic injuries, including
neuronal injury, and to developing new therapeutics for the treatment of ischemic stroke.
Acknowledgement
We thank Prof Zhong CHEN, Department of Pharma-cology, School of Medicine, Zhejiang University, for critically
reading and commenting on this manuscript.
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