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Leukotriene D4 induces brain edema and enhances CysLT2 receptor-mediated aquaporin 4 expression
BBRC Biochemical and Biophysical Research Communications 350 (2006) 399–404 www.elsevier.com/locate/ybbrc
Leukotriene D4 induces brain edema and enhances CysLT2 receptor-mediated aquaporin 4 expression Meng-Ling Wang, Xiao-Jia Huang, San-Hua Fang, Yu-Mei Yuan, Wei-Ping Zhang, Yu-Bi Lu, Qian Ding, Er-Qing Wei * Department of Pharmacology and Institute of Neuroscience, School of Medicine, Zhejiang University, 388, Yu Hang Tang Road, Hangzhou 310058, China Received 11 September 2006 Available online 20 September 2006
Abstract Cysteinyl leukotrienes (including LTC4, LTD4, and LTE4), potent inflammatory mediators, can induce brain-blood barrier (BBB) disruption and brain edema. These reactions are mediated by their receptors, CysLT1 and CysLT2 receptors. On the other hand, aquaporin 4 (AQP4) primarily modulates brain water homeostasis and edema after various injuries. Here, we aimed to determine whether AQP4 is involved in LTD4-induced brain edema. LTD4 (1 ng in 0.5 ll PBS) microinjection into the cortex increased endogenous IgG exudation (BBB disruption) and water content (brain edema), and enhanced AQP4 expression in mouse brain. The selective CysLT1 receptor antagonist pranlukast inhibited the IgG exudation, but not the increased water content and AQP4 expression induced by LTD4. In the cultured rat astrocytes, LTD4 (109–107 M, for 24 h) similarly enhanced AQP4 expression. The enhanced AQP4 expression was inhibited by Bay u9773, a non-selective CysLT1/CysLT2 receptor antagonist, but not by pranlukast. LTD4 (109–107 M) also induced the mRNA expression of CysLT2 (not CysLT1) receptor in astrocytes. These results indicate that LTD4 modulates brain edema; CysLT1 receptor mediates vasogenic edema while CysLT2 receptor may mediate cytotoxic edema via up-regulating AQP4 expression. 2006 Elsevier Inc. All rights reserved. Keywords: Leukotriene D4; Brain edema; Cysteinyl leukotriene receptor; Aquaporin 4; Astrocyte
Cysteinyl leukotrienes (CysLTs, including LTC4, LTD4, and LTE4), 5-lipoxygenase metabolites of arachidonic acid, are potent pro-inflammatory mediators [1,2]. Their actions are mediated by two G protein-coupled receptors, CysLT1 and CysLT2 receptors [1,3]. CysLTs modulate at least four responses: vascular and smooth muscle cell function, immune, inflammation, and tissue repair via activating CysLT1 and CysLT2 receptors [3]. In peripheral tissues, as an important response, CysLTs induce tissue edema in lung [4,5], liver [6], nose [7], heart [8], and kidney [9]. In the central nervous system, CysLTs induce the disruption of brain-blood barrier (BBB) and brain edema [10–13]. We have recently reported that CysLT1 receptor antagonists (pranlukast and montelukast) inhibit BBB disruption *
Corresponding author. Fax: +86 571 8820 8022. E-mail address: [email protected] (E.-Q. Wei).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.09.057
and brain edema after focal cerebral ischemia in rats [14] and mice [15], suggesting that CysLTs might mediate post-ischemic BBB disruption and the resultant brain edema via CysLT1 receptor. However, the mechanisms have not been clarified. On the other hand, it is well known that aquarorin 4 (AQP4), a member of water channel family (aquaporins), plays a major role in brain water homeostasis and brain edema [16–18]. AQP4 is primarily distributed in astrocyte endfeet surrounding the blood vessels throughout the brain and spinal cord, and in ependymal cells in ventricular surface [19]. In APQ4-deficient mice, the distinct roles of AQP4 have been proven in the two main types of brain edema, cytotoxic and vasogenic edema [17,18]. AQP4 deficiency reduces the cytotoxic edema induced by focal cerebral ischemia [18,20], water intoxication [20,21], and bacterial meningitis [22], but worsens the vasogenic (fluid
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leak) edema induced by brain tumor [17], brain abscess [23], cortical freeze-injury [17], and hydrocephalus [24]. Since both CysLTs and AQP4 in the brain are increased after focal cerebral ischemia [25,26], it is important to clarify whether CysLTs-induced post-ischemic brain edema is mediated via enhancing AQP4 expression. Therefore, in this study we determined whether exogenous LTD4, a potent agonist of both CysLT1 and CysLT2 receptors [1,3], induces brain edema and AQP4 expression in mouse brain; if so, which subtype of the receptors is involved in AQP4 expression in mouse brain and the cultured rat astrocytes. Materials and methods LTD4 microinjection. Male Kunming mice weighing 25–30 g were purchased from Shanghai Experimental Animal Center, China (Certificate No. 22-001004). Mice were anesthetized with intraperitoneal injection of 400 mg/kg chloral hydrate and immobilized on a stereotactic frame (SR-5, Narishige, Tokyo, Japan). The dura overlying the parietal cortex was exposed, and a glass micropipette (tip 40–50 lm) connected to a microinjection device was inserted into the right parietal cortex at a site 1.5 mm caudal to bregma, 4.0 mm from the midline, and 0.8 mm below the dural surface [27]. LTD4 (Sigma–Aldrich Chemicals, St. Louis, USA) 1 ng in 0.5 ll of sterile 0.1 M PBS (pH 7.4) were injected with the micropipette. The dose (1 ng) of LTD4 was found to be the most suitable one among a series of doses (0.1–100 ng) in a preliminary study. The micropipette was left in place for 10 min, to minimize back-flux of LTD4, and then removed. Mice with PBS (0.5 ll) microinjection or normal mice were used as controls. In one group, pranlukast (gifted by Dr. Masami Tsuboshima, Ono Pharmaceutical Co. Ltd., Osaka, Japan) 0.1 mg/kg, which was the most effective dose in cerebral ischemic experiments [14,15], was intraperitoneally injected 30 min before and 30 min after LTD4 microinjection. All experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Determination of brain edema and IgG exudation. To determine brain edema, the cortical samples were isolated from the injected hemispheres 24 h after microinjection, and dried at 105 C for 48 h after the wet weights were measured. Brain water content was calculated as: (wet weight–dry weight)/wet weight · 100%. To determine BBB disruption, endogenous IgG was detected by immunostaining [28] in another series. Mice were anaesthetized 24 h after microinjection and perfused transcardially with 4% paraformaldehyde after a pre-wash with saline. Brains were removed and post-fixed in 4% paraformaldehyde overnight, and then transferred to 30% sucrose for 1–3 days. Then, 8-lm coronal sections were cut by cryomicrotomy (CM1900, Leica, Germany). The sections were reacted with a biotinylated goat–anti-mouse-IgG antibody (1:500; Zhongshan, Shanghai, China) for 2 h followed by avidin–biotin-peroxidase complex (1:200; Zhongshan) for 2 h; finally, the sections were exposed for 0.5–2 min to 0.01% 3,3 0 -diaminobenzidine. IgG exudation was evaluated as the percentage increase of the gray scale in the injected region: (Gi–Gc)/Gc · 100%, where Gi is the gray scale of the injected region and Gc is that of the corresponding region in the contralateral hemisphere. Primary cultures of rat astrocytes. Astrocytes were prepared from cerebral cortices of Sprague–Dawley rats born within 24 h (Laboratory Animal Center of Zhejiang Academy of Medical Science, China) according to the methods described previously [29] with modifications. Briefly, cortical cells were trypsinized and plated onto flasks containing growth medium (high-glucose DMEM supplemented with 10% fetal bovine serum). After incubation for 12–14 days, the flasks were agitated at 260 rpm for 24 h at 37 C, and the adherent cells were trypsinized, and seeded in the growth medium. More than 95% of the cells were astrocytes as confirmed by immunocytochemical examinations with an anti-glial fibrillary acidic protein (GFAP) antibody. LTD4 (109–107 M) was
added into the culture medium in the presence or absence of pranlukast or Bay u9773 (Sigma–Aldrich Chemicals). After 24-h exposure, the AQP4 expression in astrocytes was determined. Determination of AQP4 expression. To determine AQP4 expression by immunostaining, brain sections or the astrocytes cultured on coverslips (fixed by cold methanol at 10 C for 5 min) were sequentially incubated with goat serum (1:20) for 2 h, a rabbit anti-AQP4 affinity-purified polyclonal antibody (1:150; Chemicon International, Temecula, CA, USA) at 4 C overnight, and TRITC-labeled goat–anti-rabbit IgG (1:200; Chemicon) at room temperature for 2 h. Finally, the brain sections and the astrocytes were examined under a fluorescent microscope (Nikon Eclipse TE2000-E, Japan). AQP4 protein expression was determined by Western blot analysis. Briefly, protein samples (50 lg) from brains or astrocytes were separated by 12% SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were reacted with a rabbit polyclonal antibody against AQP4 (1:500) and a mouse monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000, Kangchen Biotechnology Inc., Shanghai, China) at 4 C overnight. Following repeated washes, the membranes were sequentially reacted with a horseradish peroxidase-conjugated goat–anti-rabbit antibody (1:200; Zhongshan, Shanghai, China), at room temperature for 2 h, and the ECL reagents; finally exposed on an X-ray film to show the AQP4 band (34.8 kDa). After being thoroughly washed, the membranes were then reacted with another secondary antibody, a horseradish peroxidaseconjugated goat–anti-mouse IgG (1:2000; Chemicon), to show the GAPDH band (36 kDa). The optical densities were quantitatively analyzed with a laser densitometer (UltroScan XL, Pharmacia LKB Co., Sweden). The results of AQP4 expression are reported as the percentage changes over GAPDH. Reverse transcription-polymerase chain reaction (RT-PCR). To determine the mRNA expressions of CysLT1 and CysLT2 receptors, total RNA was extracted from astrocytes or a rat brain using Trizol reagents (Invitrogen, USA) according to the manufacturer’s protocol. The cDNA synthesis and PCRs were performed as reported [30]. The primer sequences were as the following: rat CysLT1 receptor forward 5 0 -(+) TCT CCG TTG TGG GTT TCT-3 0 and reverse 5 0 -(+) TAT AAG GCA TAG GTG GTG-3 0 (product size 214 bp); rat CysLT2 receptor forward 5 0 -(+) AGC GTT AGG AGT GCC TGG AT-3 0 and reverse 5 0 -(+) CAA GTG GAT GGT CCG AAG TG-3 0 (product size 520 bp); b-actin forward 5 0 -(+) TAC AAC CTC CTT GCA GCT CC-3 0 and reverse 5 0 -(+) GGA TCT TCA TGA GGT AGT CAG TC-3 0 (product size 620 bp), or forward 5 0 -(+) AAC CCT AAG GCC AAC CGT GAA-3 0 , and reverse 5 0 -(+) TCA TGA GGT AGT CTG TCA GGT C-3 0 (product size 285 bp). Statistical analysis. Data are reported as means ± SD. Statistical analyses were performed using one-way ANOVA with Newman–Keuls Post Hoc Multiple Comparison (SPSS 10.0 for Windows, 1999, SPSS Inc., USA). A value of P < 0.05 was considered statistically significant.
Results BBB disruption and brain edema LTD4 (1 ng)-induced endogenous IgG exudation from 6 to 72 h after microinjection with a peak at 24 h (data not shown) indicating BBB disruption. IgG was leaked to the injected region (upper panels in Fig. 1A) and localized around the vessels and extravascular tissue (lower panels in Fig. 1A) 24 h after LTD4 microinjection. Pranlukast (0.1 mg/kg) ameliorated LTD4-induced IgG exudation (Fig. 1B). Otherwise, LTD4 increased the water content in the injected cortex indicating brain edema; however, pranlukast did not reduce the increased water content (Fig. 1C).
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and B) and AQP4 protein expression (Fig. 2C) 24 h after microinjection. Pranlukast did not affect LTD4-induced AQP4 expression (Fig. 2A–C). In the cultured rat astrocytes, LTD4 increased AQP4 expression at concentrations of 109–107 M (Fig. 3A and B). Pranlukast (109– 106 M) had no significant effect on LTD4 (108 M)-increased AQP4 expression; however, Bay u9773 (109– 107 M) did concentration-dependently reduce the expression (Fig. 3A and C). Pranlukast or Bay u9773 alone did not change AQP4 immunoreactivity (lower panels in Fig. 3A). CysLT1 and CysLT2 receptor mRNA expressions in astrocytes
Fig. 1. Endogenous IgG exudation and water content in the cortex 24 h after LTD4 microinjection in mice. (A) IgG exudation is localized in the LTD4 (1 ng)-injected sites (upper panels) around the vessels and extravascular tissue (lower panels). (B) LTD4-induced IgG exudation was ameliorated by pranlukast (0.1 mg/kg). (C) LTD4 also increased water content; pranlukast did not inhibit the increased water content. Data are summarized as means ± SD; n = 8 mice for each group; *P < 0.05 and **P < 0.01 compared with normal group, #P < 0.05 compared with LTD , 4 one-way ANOVA. Scale bars in (A) = 200 lm (upper panels) or 50 lm (lower panels).
AQP4 expression In mouse brain, LTD4 (1 ng) significantly increased the number of AQP4-positive cells (microvessels) (Fig. 2A
Fig. 2. AQP4 expression in the cortex 24 h after LTD4 microinjection in mice. (A) AQP4 immunoreactivity and (B) the number of APQ4-positive cells are increased in ipsilateral cortices after LTD4 (1 ng) microinjection. (C) The increased expression of AQP4 protein was also detected by Western blot analysis. Pranlukast (0.1 mg/kg) did not affect AQP4 expression (A–C). Data are summarized as means ± SD; n = 8 mice (immunostaining) or 5 mice (Western blot) for each group; *P < 0.05 compared with normal group, one-way ANOVA. Scale bar = 50 lm.
CysLT1 receptor mRNA was expressed in the astrocytes as similar to rat brain and was not significantly increased after 24-h exposure to LTD4 109–107 M (Fig. 4A). However, the expression of CysLT2 receptor mRNA was much weaker in the astrocytes than in the brain. LTD4 109– 107 M significantly increased the expression of CysLT2 receptor mRNA in the astrocytes (Fig. 4B). Discussion In the present study, we found that LTD4 induces brain edema, which is associated with BBB disruption and the up-regulation of AQP4 expression, and that CysLT2, rather than CysLT1, receptor may be involved in the up-regulation of AQP4 expression. Our findings provide direct evidence for the CysLTs-induced brain edema as previously reported [10–13], and further reveal that this type of brain edema may be partly modulated via AQP4. LTD4-induced brain edema seems to include vasogenic and cytotoxic edemas. Vasogenic edema occurs when BBB is disrupted, which permits plasma fluid into the brain extravascular space; while cytotoxic edema occurs when water flows from the vascular compartment through intact BBB and astrocytic foot processes, and accumulates primarily in astrocytes [16]. In the present study, LTD4induced BBB disruption is evidenced by endogenous IgG exudation in mouse brain, which is attenuated by the selective CysLT1 receptor antagonist pranlukast (Fig. 1A and B). Thus, we propose that LTD4 might induce vasogenic edema via CysLT1 receptor-mediated BBB disruption. This action is supported by the localization of CysLT1 receptor in brain microvascular endothelial cells as reported in human and rat brain [31,32]. However, pranlukast did not inhibit LTD4-induced increase in brain water content (Fig. 1C) and AQP4 expression (Fig. 2), indicating that it does not inhibit AQP4-related brain edema. AQP4 promotes cytotoxic edema and attenuates vasogenic edema [16,18], so that LTD4 might induce cytotoxic edema by enhancing AQP4 expression (Fig. 2). This response may not be mediated via CysLT1 receptor. Similarly, LTD4 concentration-dependently enhanced AQP4 expression in astrocytes (Fig. 3). The LTD4-
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Fig. 3. AQP4 expression after 24-h exposure to LTD4 in the cultured rat astrocytes. (A) LTD4 (109–107 M) increases astrocyte AQP4 immunoreactivity (red, upper panels). LTD4 (108 M)-increased immunoreactivity is inhibited by Bay u9773 107 M but not by pranlukast 106 M; while pranlukast or Bay u9773 alone does not affect AQP4 immunoreactivity (lower panels). The nuclei are stained by DAPI (blue). (B) LTD4-increased AQP4 protein expression was confirmed by Western blot analysis. (C) Pranlukast (109–106 M) did not inhibit but Bay u9773 (109–107 M) inhibited LTD4 (108 M)-increased AQP4 expression. Data are summarized as means ± SD; n = 4 for each group; *P < 0.05 and **P < 0.01 compared with normal control, #P < 0.05 and ## P < 0.01 compared with LTD4, one-way ANOVA. 1, normal control; 2, LTD4 108 M; 3–6, LTD4 + pranlukast 109, 108, 107, and 106 M; 7–10, LTD4 + Bay u9773 1010, 109, 108, and 107 M. Scale bars = 50 lm.
Fig. 4. mRNA expression of CysLT1 (A) or CysLT2 receptor (B) after 24-h exposure to LTD4 in the cultured rat astrocytes. RT-PCR analysis shows that LTD4 109–107 M did not increase astrocyte CysLT1 receptor mRNA (214 bp) expression (A), but did increase CysLT2 receptor mRNA (520 bp) expression (B). Sample from normal rat brain was used as the control. Data are summarized as means ± SD; n = 4 for each group; **P < 0.01 compared with normal astrocytes without LTD4, one-way ANOVA. M, marker; 1, normal astrocytes; 2–4, astrocytes treated with LTD4 109, 108, and 107 M; 5, normal rat brain.
enhanced AQP4 expression was not affected by pranlukast but inhibited by Bay u9773, a non-selective CysLT1/CysLT2 receptor antagonist [33]. Because no selective CysLT2 receptor antagonists are currently available, the effect of Bay u9773 may represent the blockade of CysLT2 receptor
in comparison with the effect of pranlukast. Furthermore, we found that the mRNA expression of CysLT2 receptor was weak in normal astrocytes but significantly increased by LTD4; while the relatively higher mRNA expression of CysLT1 receptor was not further increased by LTD4
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(Fig. 4). The mRNA expression of CysLT2 receptor was also found to be much weaker than that of CysLT1 receptor in rat astrocytes [34]. Together with the results of the receptor antagonism and expression, LTD4-induced AQP4 expression may be mediated by CysLT2 receptor activation. However, we have previously reported that pranlukast attenuated both BBB disruption and brain edema after focal cerebral ischemia in rats and mice [14,15]. Since the contents of CysLTs in the brain are increased after ischemia [25], the effect of exogenous LTD4 partly mimics the post-ischemic pathological changes. The effect of pranlukast on LTD4-induced BBB disruption is consistent with that we previously reported, but its effect on brain edema (increase in water content) is different. One of the possibilities for the difference is that inhibition of vasogenic edema by pranlukast may partially reduce post-ischemic edema. Another possibility is that ischemia-induced brain edema results from much more complex responses. Therefore, other anti-inflammatory abilities of pranlukast, such as inhibition of inflammatory cell infiltration [35,36] and cytokine production [37], may be also involved in the effect on post-ischemic edema. The present results suggest that CysLT1 receptor antagonist(s) may not substantially attenuate the AQP4-related cytotoxic edema. In summary, we found that LTD4 modulates brain edema; CysLT1 receptor mediates vasogenic edema while CysLT2 receptor may mediate cytotoxic edema via up-regulating AQP4 expression. These findings indicate the distinct roles of CysLT1 and CysLT2 receptors in the pathophysiological processes in the brain. Acknowledgments This study was supported by the National Natural Science Foundation of China (30500613) and the Scientific Foundation of Education Ministry of China (20050335105). We thank Dr. Masami Tsuboshima, Ono Pharmaceutical Co., Japan, for the supply of pranlukast. References [1] C. Brink, S.E. Dahlen, J. Drazen, J.F. Evans, D.W. Hay, S. Nicosia, C.N. Serhan, T. Shimizu, T. Yokomizo, International Union of Pharmacology XXXVII. Nomenclature for leukotriene and lipoxin receptors, Pharmacol. Rev. 55 (2003) 195–227. [2] C.D. Funk, Prostaglandins and leukotrienes: advances in eicosanoid biology, Science 294 (2001) 1871–1875. [3] Y. Kanaoka, J.A. Boyce, Cysteinyl leukotrienes and their receptors: cellular distribution and function in immune and inflammatory responses, J. Immunol. 173 (2004) 1503–1510. [4] K.S. Lee, S.R. Kim, H.S. Park, G.Y. Jin, Y.C. Lee, Cysteinyl leukotriene receptor antagonist regulates vascular permeability by reducing vascular endothelial growth factor expression, J. Allergy Clin. Immunol. 114 (2004) 1093–1099. [5] D.E. Sloniewsky, K.M. Ridge, Y. Adir, F.P. Fries, A. Briva, J.I. Sznajder, P.H. Sporn, Leukotriene D4 activates alveolar epithelial Na, K-ATPase and increases alveolar fluid clearance, Am. J. Respir. Crit. Care Med. 169 (2004) 407–412.
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Leukotriene D4 induces brain edema and enhances CysLT2 receptor-mediated aquaporin 4 expression Meng-Ling Wang, Xiao-Jia Huang, San-Hua Fang, Yu-Mei Yuan, Wei-Ping Zhang, Yu-Bi Lu, Qian Ding, Er-Qing Wei * Department of Pharmacology and Institute of Neuroscience, School of Medicine, Zhejiang University, 388, Yu Hang Tang Road, Hangzhou 310058, China Received 11 September 2006 Available online 20 September 2006
Abstract Cysteinyl leukotrienes (including LTC4, LTD4, and LTE4), potent inflammatory mediators, can induce brain-blood barrier (BBB) disruption and brain edema. These reactions are mediated by their receptors, CysLT1 and CysLT2 receptors. On the other hand, aquaporin 4 (AQP4) primarily modulates brain water homeostasis and edema after various injuries. Here, we aimed to determine whether AQP4 is involved in LTD4-induced brain edema. LTD4 (1 ng in 0.5 ll PBS) microinjection into the cortex increased endogenous IgG exudation (BBB disruption) and water content (brain edema), and enhanced AQP4 expression in mouse brain. The selective CysLT1 receptor antagonist pranlukast inhibited the IgG exudation, but not the increased water content and AQP4 expression induced by LTD4. In the cultured rat astrocytes, LTD4 (109–107 M, for 24 h) similarly enhanced AQP4 expression. The enhanced AQP4 expression was inhibited by Bay u9773, a non-selective CysLT1/CysLT2 receptor antagonist, but not by pranlukast. LTD4 (109–107 M) also induced the mRNA expression of CysLT2 (not CysLT1) receptor in astrocytes. These results indicate that LTD4 modulates brain edema; CysLT1 receptor mediates vasogenic edema while CysLT2 receptor may mediate cytotoxic edema via up-regulating AQP4 expression. 2006 Elsevier Inc. All rights reserved. Keywords: Leukotriene D4; Brain edema; Cysteinyl leukotriene receptor; Aquaporin 4; Astrocyte
Cysteinyl leukotrienes (CysLTs, including LTC4, LTD4, and LTE4), 5-lipoxygenase metabolites of arachidonic acid, are potent pro-inflammatory mediators [1,2]. Their actions are mediated by two G protein-coupled receptors, CysLT1 and CysLT2 receptors [1,3]. CysLTs modulate at least four responses: vascular and smooth muscle cell function, immune, inflammation, and tissue repair via activating CysLT1 and CysLT2 receptors [3]. In peripheral tissues, as an important response, CysLTs induce tissue edema in lung [4,5], liver [6], nose [7], heart [8], and kidney [9]. In the central nervous system, CysLTs induce the disruption of brain-blood barrier (BBB) and brain edema [10–13]. We have recently reported that CysLT1 receptor antagonists (pranlukast and montelukast) inhibit BBB disruption *
Corresponding author. Fax: +86 571 8820 8022. E-mail address: [email protected] (E.-Q. Wei).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.09.057
and brain edema after focal cerebral ischemia in rats [14] and mice [15], suggesting that CysLTs might mediate post-ischemic BBB disruption and the resultant brain edema via CysLT1 receptor. However, the mechanisms have not been clarified. On the other hand, it is well known that aquarorin 4 (AQP4), a member of water channel family (aquaporins), plays a major role in brain water homeostasis and brain edema [16–18]. AQP4 is primarily distributed in astrocyte endfeet surrounding the blood vessels throughout the brain and spinal cord, and in ependymal cells in ventricular surface [19]. In APQ4-deficient mice, the distinct roles of AQP4 have been proven in the two main types of brain edema, cytotoxic and vasogenic edema [17,18]. AQP4 deficiency reduces the cytotoxic edema induced by focal cerebral ischemia [18,20], water intoxication [20,21], and bacterial meningitis [22], but worsens the vasogenic (fluid
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leak) edema induced by brain tumor [17], brain abscess [23], cortical freeze-injury [17], and hydrocephalus [24]. Since both CysLTs and AQP4 in the brain are increased after focal cerebral ischemia [25,26], it is important to clarify whether CysLTs-induced post-ischemic brain edema is mediated via enhancing AQP4 expression. Therefore, in this study we determined whether exogenous LTD4, a potent agonist of both CysLT1 and CysLT2 receptors [1,3], induces brain edema and AQP4 expression in mouse brain; if so, which subtype of the receptors is involved in AQP4 expression in mouse brain and the cultured rat astrocytes. Materials and methods LTD4 microinjection. Male Kunming mice weighing 25–30 g were purchased from Shanghai Experimental Animal Center, China (Certificate No. 22-001004). Mice were anesthetized with intraperitoneal injection of 400 mg/kg chloral hydrate and immobilized on a stereotactic frame (SR-5, Narishige, Tokyo, Japan). The dura overlying the parietal cortex was exposed, and a glass micropipette (tip 40–50 lm) connected to a microinjection device was inserted into the right parietal cortex at a site 1.5 mm caudal to bregma, 4.0 mm from the midline, and 0.8 mm below the dural surface [27]. LTD4 (Sigma–Aldrich Chemicals, St. Louis, USA) 1 ng in 0.5 ll of sterile 0.1 M PBS (pH 7.4) were injected with the micropipette. The dose (1 ng) of LTD4 was found to be the most suitable one among a series of doses (0.1–100 ng) in a preliminary study. The micropipette was left in place for 10 min, to minimize back-flux of LTD4, and then removed. Mice with PBS (0.5 ll) microinjection or normal mice were used as controls. In one group, pranlukast (gifted by Dr. Masami Tsuboshima, Ono Pharmaceutical Co. Ltd., Osaka, Japan) 0.1 mg/kg, which was the most effective dose in cerebral ischemic experiments [14,15], was intraperitoneally injected 30 min before and 30 min after LTD4 microinjection. All experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Determination of brain edema and IgG exudation. To determine brain edema, the cortical samples were isolated from the injected hemispheres 24 h after microinjection, and dried at 105 C for 48 h after the wet weights were measured. Brain water content was calculated as: (wet weight–dry weight)/wet weight · 100%. To determine BBB disruption, endogenous IgG was detected by immunostaining [28] in another series. Mice were anaesthetized 24 h after microinjection and perfused transcardially with 4% paraformaldehyde after a pre-wash with saline. Brains were removed and post-fixed in 4% paraformaldehyde overnight, and then transferred to 30% sucrose for 1–3 days. Then, 8-lm coronal sections were cut by cryomicrotomy (CM1900, Leica, Germany). The sections were reacted with a biotinylated goat–anti-mouse-IgG antibody (1:500; Zhongshan, Shanghai, China) for 2 h followed by avidin–biotin-peroxidase complex (1:200; Zhongshan) for 2 h; finally, the sections were exposed for 0.5–2 min to 0.01% 3,3 0 -diaminobenzidine. IgG exudation was evaluated as the percentage increase of the gray scale in the injected region: (Gi–Gc)/Gc · 100%, where Gi is the gray scale of the injected region and Gc is that of the corresponding region in the contralateral hemisphere. Primary cultures of rat astrocytes. Astrocytes were prepared from cerebral cortices of Sprague–Dawley rats born within 24 h (Laboratory Animal Center of Zhejiang Academy of Medical Science, China) according to the methods described previously [29] with modifications. Briefly, cortical cells were trypsinized and plated onto flasks containing growth medium (high-glucose DMEM supplemented with 10% fetal bovine serum). After incubation for 12–14 days, the flasks were agitated at 260 rpm for 24 h at 37 C, and the adherent cells were trypsinized, and seeded in the growth medium. More than 95% of the cells were astrocytes as confirmed by immunocytochemical examinations with an anti-glial fibrillary acidic protein (GFAP) antibody. LTD4 (109–107 M) was
added into the culture medium in the presence or absence of pranlukast or Bay u9773 (Sigma–Aldrich Chemicals). After 24-h exposure, the AQP4 expression in astrocytes was determined. Determination of AQP4 expression. To determine AQP4 expression by immunostaining, brain sections or the astrocytes cultured on coverslips (fixed by cold methanol at 10 C for 5 min) were sequentially incubated with goat serum (1:20) for 2 h, a rabbit anti-AQP4 affinity-purified polyclonal antibody (1:150; Chemicon International, Temecula, CA, USA) at 4 C overnight, and TRITC-labeled goat–anti-rabbit IgG (1:200; Chemicon) at room temperature for 2 h. Finally, the brain sections and the astrocytes were examined under a fluorescent microscope (Nikon Eclipse TE2000-E, Japan). AQP4 protein expression was determined by Western blot analysis. Briefly, protein samples (50 lg) from brains or astrocytes were separated by 12% SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were reacted with a rabbit polyclonal antibody against AQP4 (1:500) and a mouse monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000, Kangchen Biotechnology Inc., Shanghai, China) at 4 C overnight. Following repeated washes, the membranes were sequentially reacted with a horseradish peroxidase-conjugated goat–anti-rabbit antibody (1:200; Zhongshan, Shanghai, China), at room temperature for 2 h, and the ECL reagents; finally exposed on an X-ray film to show the AQP4 band (34.8 kDa). After being thoroughly washed, the membranes were then reacted with another secondary antibody, a horseradish peroxidaseconjugated goat–anti-mouse IgG (1:2000; Chemicon), to show the GAPDH band (36 kDa). The optical densities were quantitatively analyzed with a laser densitometer (UltroScan XL, Pharmacia LKB Co., Sweden). The results of AQP4 expression are reported as the percentage changes over GAPDH. Reverse transcription-polymerase chain reaction (RT-PCR). To determine the mRNA expressions of CysLT1 and CysLT2 receptors, total RNA was extracted from astrocytes or a rat brain using Trizol reagents (Invitrogen, USA) according to the manufacturer’s protocol. The cDNA synthesis and PCRs were performed as reported [30]. The primer sequences were as the following: rat CysLT1 receptor forward 5 0 -(+) TCT CCG TTG TGG GTT TCT-3 0 and reverse 5 0 -(+) TAT AAG GCA TAG GTG GTG-3 0 (product size 214 bp); rat CysLT2 receptor forward 5 0 -(+) AGC GTT AGG AGT GCC TGG AT-3 0 and reverse 5 0 -(+) CAA GTG GAT GGT CCG AAG TG-3 0 (product size 520 bp); b-actin forward 5 0 -(+) TAC AAC CTC CTT GCA GCT CC-3 0 and reverse 5 0 -(+) GGA TCT TCA TGA GGT AGT CAG TC-3 0 (product size 620 bp), or forward 5 0 -(+) AAC CCT AAG GCC AAC CGT GAA-3 0 , and reverse 5 0 -(+) TCA TGA GGT AGT CTG TCA GGT C-3 0 (product size 285 bp). Statistical analysis. Data are reported as means ± SD. Statistical analyses were performed using one-way ANOVA with Newman–Keuls Post Hoc Multiple Comparison (SPSS 10.0 for Windows, 1999, SPSS Inc., USA). A value of P < 0.05 was considered statistically significant.
Results BBB disruption and brain edema LTD4 (1 ng)-induced endogenous IgG exudation from 6 to 72 h after microinjection with a peak at 24 h (data not shown) indicating BBB disruption. IgG was leaked to the injected region (upper panels in Fig. 1A) and localized around the vessels and extravascular tissue (lower panels in Fig. 1A) 24 h after LTD4 microinjection. Pranlukast (0.1 mg/kg) ameliorated LTD4-induced IgG exudation (Fig. 1B). Otherwise, LTD4 increased the water content in the injected cortex indicating brain edema; however, pranlukast did not reduce the increased water content (Fig. 1C).
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and B) and AQP4 protein expression (Fig. 2C) 24 h after microinjection. Pranlukast did not affect LTD4-induced AQP4 expression (Fig. 2A–C). In the cultured rat astrocytes, LTD4 increased AQP4 expression at concentrations of 109–107 M (Fig. 3A and B). Pranlukast (109– 106 M) had no significant effect on LTD4 (108 M)-increased AQP4 expression; however, Bay u9773 (109– 107 M) did concentration-dependently reduce the expression (Fig. 3A and C). Pranlukast or Bay u9773 alone did not change AQP4 immunoreactivity (lower panels in Fig. 3A). CysLT1 and CysLT2 receptor mRNA expressions in astrocytes
Fig. 1. Endogenous IgG exudation and water content in the cortex 24 h after LTD4 microinjection in mice. (A) IgG exudation is localized in the LTD4 (1 ng)-injected sites (upper panels) around the vessels and extravascular tissue (lower panels). (B) LTD4-induced IgG exudation was ameliorated by pranlukast (0.1 mg/kg). (C) LTD4 also increased water content; pranlukast did not inhibit the increased water content. Data are summarized as means ± SD; n = 8 mice for each group; *P < 0.05 and **P < 0.01 compared with normal group, #P < 0.05 compared with LTD , 4 one-way ANOVA. Scale bars in (A) = 200 lm (upper panels) or 50 lm (lower panels).
AQP4 expression In mouse brain, LTD4 (1 ng) significantly increased the number of AQP4-positive cells (microvessels) (Fig. 2A
Fig. 2. AQP4 expression in the cortex 24 h after LTD4 microinjection in mice. (A) AQP4 immunoreactivity and (B) the number of APQ4-positive cells are increased in ipsilateral cortices after LTD4 (1 ng) microinjection. (C) The increased expression of AQP4 protein was also detected by Western blot analysis. Pranlukast (0.1 mg/kg) did not affect AQP4 expression (A–C). Data are summarized as means ± SD; n = 8 mice (immunostaining) or 5 mice (Western blot) for each group; *P < 0.05 compared with normal group, one-way ANOVA. Scale bar = 50 lm.
CysLT1 receptor mRNA was expressed in the astrocytes as similar to rat brain and was not significantly increased after 24-h exposure to LTD4 109–107 M (Fig. 4A). However, the expression of CysLT2 receptor mRNA was much weaker in the astrocytes than in the brain. LTD4 109– 107 M significantly increased the expression of CysLT2 receptor mRNA in the astrocytes (Fig. 4B). Discussion In the present study, we found that LTD4 induces brain edema, which is associated with BBB disruption and the up-regulation of AQP4 expression, and that CysLT2, rather than CysLT1, receptor may be involved in the up-regulation of AQP4 expression. Our findings provide direct evidence for the CysLTs-induced brain edema as previously reported [10–13], and further reveal that this type of brain edema may be partly modulated via AQP4. LTD4-induced brain edema seems to include vasogenic and cytotoxic edemas. Vasogenic edema occurs when BBB is disrupted, which permits plasma fluid into the brain extravascular space; while cytotoxic edema occurs when water flows from the vascular compartment through intact BBB and astrocytic foot processes, and accumulates primarily in astrocytes [16]. In the present study, LTD4induced BBB disruption is evidenced by endogenous IgG exudation in mouse brain, which is attenuated by the selective CysLT1 receptor antagonist pranlukast (Fig. 1A and B). Thus, we propose that LTD4 might induce vasogenic edema via CysLT1 receptor-mediated BBB disruption. This action is supported by the localization of CysLT1 receptor in brain microvascular endothelial cells as reported in human and rat brain [31,32]. However, pranlukast did not inhibit LTD4-induced increase in brain water content (Fig. 1C) and AQP4 expression (Fig. 2), indicating that it does not inhibit AQP4-related brain edema. AQP4 promotes cytotoxic edema and attenuates vasogenic edema [16,18], so that LTD4 might induce cytotoxic edema by enhancing AQP4 expression (Fig. 2). This response may not be mediated via CysLT1 receptor. Similarly, LTD4 concentration-dependently enhanced AQP4 expression in astrocytes (Fig. 3). The LTD4-
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Fig. 3. AQP4 expression after 24-h exposure to LTD4 in the cultured rat astrocytes. (A) LTD4 (109–107 M) increases astrocyte AQP4 immunoreactivity (red, upper panels). LTD4 (108 M)-increased immunoreactivity is inhibited by Bay u9773 107 M but not by pranlukast 106 M; while pranlukast or Bay u9773 alone does not affect AQP4 immunoreactivity (lower panels). The nuclei are stained by DAPI (blue). (B) LTD4-increased AQP4 protein expression was confirmed by Western blot analysis. (C) Pranlukast (109–106 M) did not inhibit but Bay u9773 (109–107 M) inhibited LTD4 (108 M)-increased AQP4 expression. Data are summarized as means ± SD; n = 4 for each group; *P < 0.05 and **P < 0.01 compared with normal control, #P < 0.05 and ## P < 0.01 compared with LTD4, one-way ANOVA. 1, normal control; 2, LTD4 108 M; 3–6, LTD4 + pranlukast 109, 108, 107, and 106 M; 7–10, LTD4 + Bay u9773 1010, 109, 108, and 107 M. Scale bars = 50 lm.
Fig. 4. mRNA expression of CysLT1 (A) or CysLT2 receptor (B) after 24-h exposure to LTD4 in the cultured rat astrocytes. RT-PCR analysis shows that LTD4 109–107 M did not increase astrocyte CysLT1 receptor mRNA (214 bp) expression (A), but did increase CysLT2 receptor mRNA (520 bp) expression (B). Sample from normal rat brain was used as the control. Data are summarized as means ± SD; n = 4 for each group; **P < 0.01 compared with normal astrocytes without LTD4, one-way ANOVA. M, marker; 1, normal astrocytes; 2–4, astrocytes treated with LTD4 109, 108, and 107 M; 5, normal rat brain.
enhanced AQP4 expression was not affected by pranlukast but inhibited by Bay u9773, a non-selective CysLT1/CysLT2 receptor antagonist [33]. Because no selective CysLT2 receptor antagonists are currently available, the effect of Bay u9773 may represent the blockade of CysLT2 receptor
in comparison with the effect of pranlukast. Furthermore, we found that the mRNA expression of CysLT2 receptor was weak in normal astrocytes but significantly increased by LTD4; while the relatively higher mRNA expression of CysLT1 receptor was not further increased by LTD4
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(Fig. 4). The mRNA expression of CysLT2 receptor was also found to be much weaker than that of CysLT1 receptor in rat astrocytes [34]. Together with the results of the receptor antagonism and expression, LTD4-induced AQP4 expression may be mediated by CysLT2 receptor activation. However, we have previously reported that pranlukast attenuated both BBB disruption and brain edema after focal cerebral ischemia in rats and mice [14,15]. Since the contents of CysLTs in the brain are increased after ischemia [25], the effect of exogenous LTD4 partly mimics the post-ischemic pathological changes. The effect of pranlukast on LTD4-induced BBB disruption is consistent with that we previously reported, but its effect on brain edema (increase in water content) is different. One of the possibilities for the difference is that inhibition of vasogenic edema by pranlukast may partially reduce post-ischemic edema. Another possibility is that ischemia-induced brain edema results from much more complex responses. Therefore, other anti-inflammatory abilities of pranlukast, such as inhibition of inflammatory cell infiltration [35,36] and cytokine production [37], may be also involved in the effect on post-ischemic edema. The present results suggest that CysLT1 receptor antagonist(s) may not substantially attenuate the AQP4-related cytotoxic edema. In summary, we found that LTD4 modulates brain edema; CysLT1 receptor mediates vasogenic edema while CysLT2 receptor may mediate cytotoxic edema via up-regulating AQP4 expression. These findings indicate the distinct roles of CysLT1 and CysLT2 receptors in the pathophysiological processes in the brain. Acknowledgments This study was supported by the National Natural Science Foundation of China (30500613) and the Scientific Foundation of Education Ministry of China (20050335105). We thank Dr. Masami Tsuboshima, Ono Pharmaceutical Co., Japan, for the supply of pranlukast. References [1] C. Brink, S.E. Dahlen, J. Drazen, J.F. Evans, D.W. Hay, S. Nicosia, C.N. Serhan, T. Shimizu, T. Yokomizo, International Union of Pharmacology XXXVII. Nomenclature for leukotriene and lipoxin receptors, Pharmacol. Rev. 55 (2003) 195–227. [2] C.D. Funk, Prostaglandins and leukotrienes: advances in eicosanoid biology, Science 294 (2001) 1871–1875. [3] Y. Kanaoka, J.A. Boyce, Cysteinyl leukotrienes and their receptors: cellular distribution and function in immune and inflammatory responses, J. Immunol. 173 (2004) 1503–1510. [4] K.S. Lee, S.R. Kim, H.S. Park, G.Y. Jin, Y.C. Lee, Cysteinyl leukotriene receptor antagonist regulates vascular permeability by reducing vascular endothelial growth factor expression, J. Allergy Clin. Immunol. 114 (2004) 1093–1099. [5] D.E. Sloniewsky, K.M. Ridge, Y. Adir, F.P. Fries, A. Briva, J.I. Sznajder, P.H. Sporn, Leukotriene D4 activates alveolar epithelial Na, K-ATPase and increases alveolar fluid clearance, Am. J. Respir. Crit. Care Med. 169 (2004) 407–412.
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