On the other hand it is

On the other hand, it is well known that aquarorin 4 (AQP4), a member of water channel family (aquaporins), plays a major role in PPADS tetrasodium salt receptor water homeostasis and brain edema , , . AQP4 is primarily distributed in astrocyte endfeet surrounding the blood vessels throughout the brain and spinal cord, and in ependymal cells in ventricular surface . In APQ4-deficient mice, the distinct roles of AQP4 have been proven in the two main types of brain edema, cytotoxic and vasogenic edema , . AQP4 deficiency reduces the cytotoxic edema induced by focal cerebral ischemia , , water intoxication , , and bacterial meningitis , but worsens the vasogenic (fluid leak) edema induced by brain tumor , brain abscess , cortical freeze-injury , and hydrocephalus . Since both CysLTs and AQP4 in the brain are increased after focal cerebral ischemia , , 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 LTD, a potent agonist of both CysLT and CysLT receptors , , 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 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 [27]. 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 [14], [15], 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 [28] 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.