Therefore in this study we determined whether exogenous LTD
Therefore, in this study we determined whether exogenous LTD, a potent agonist of both CysLT and CysLT receptors , , induces ascorbic acid sale 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 LTD4microinjection. Male Kunming mice weighing 25–30g were purchased from Shanghai Experimental Animal Center, China (Certificate No. 22-001004). Mice were anesthetized with intraperitoneal injection of 400mg/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 40–50μm) connected to a microinjection device was inserted into the right parietal cortex at a site 1.5mm caudal to bregma, 4.0mm from the midline, and 0.8mm below the dural surface . LTD4 (Sigma–Aldrich Chemicals, St. Louis, USA) 1ng in 0.5μl of sterile 0.1M PBS (pH 7.4) were injected with the micropipette. The dose (1ng) of LTD4 was found to be the most suitable one among a series of doses (0.1–100ng) in a preliminary study. The micropipette was left in place for 10min, to minimize back-flux of LTD4, and then removed. Mice with PBS (0.5μl) microinjection or normal mice were used as controls. In one group, pranlukast (gifted by Dr. Masami Tsuboshima, Ono Pharmaceutical Co. Ltd., Osaka, Japan) 0.1mg/kg, which was the most effective dose in cerebral ischemic experiments , , was intraperitoneally injected 30min before and 30min 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 24h after microinjection, and dried at 105°C for 48h 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  in another series. Mice were anaesthetized 24h 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-μm 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 2h followed by avidin–biotin-peroxidase complex (1:200; Zhongshan) for 2h; finally, the sections were exposed for 0.5–2min to 0.01% 3,3′-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 24h (Laboratory Animal Center of Zhejiang Academy of Medical Science, China) according to the methods described previously  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 260rpm for 24h 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 (10−9–10−7M) 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 5min) were sequentially incubated with goat serum (1:20) for 2h, 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 2h. Finally, the brain sections and the astrocytes were examined under a fluorescent microscope (Nikon Eclipse TE2000-E, Japan).