The samples were then resolved by SDS polyacrylamide gel electrophoresis

usion pump for 4 hr before/after SE induction. Microdialysis samples were collected 240 l before and after SE induction, respectively. Samples were transferred to -80 C freezer and stored until analysis. At the end of the experimental period, the animals were killed, and their brains were fixed to identify the location of the microdialysis probe in the PC. Only the animals with the probe tip in the designated location were included in the analysis. Surgery For microdialysis, rats were anesthetized and placed in a stereotaxic frame. Thereafter, a guide cannula was implanted in the PC, according to the rat brain atlas. Rats were allowed 7 days to recover from the surgical procedure before the start of microdialysis. Animals were divided into five groups for intracerebroventricular drug infusion: vehicle, sTNFp55R, SN50 BQ-788, Cav1, and one group for intraperitoneal injection of apocynin. The dosage of each compound did 2181489 not affect seizure threshold, seizure score, mortality during SE, and BBB integrity in non-SE animals in the preliminary study. Animals were anesthetized and placed in a stereotaxic frame. For the osmotic pump implantation, holes were drilled through the skull for introducing a brain infusion kit 1 into the right lateral ventricle, according to the atlas. The infusion kit was sealed with dental cement and connected to an osmotic pump. The pump was placed in a subcutaneous pocket in the dorsal region. Animals received 0.5 l/h of vehicle or compound for 1 week. The compounds began to be immediately infused after surgery. Rats were allowed 3 days to recover from the surgical procedure before SE induction. Because the volume of vasogenic edema peaked at 2-3 days after SE in our previous studies, our experimental schedules inhibited the function of the related molecules from 3 Enzyme-linked immunosorbent assay and nitric oxide assay The concentration of TNF-, and NO in perfusates were measured using the Quantikine ELISA kits, and a nitrate/nitrite fluorometric assay kit, according to the manufacturer’s instructions. The concentration of big ET-1 in perfusates was measured using the rat big ET-1 ELISA kit, according to the manufacturer’s instructions. Big ET-1 is processed to ET-1. Big ET-1 is more stable than ET-1. However, all ET-1 assay kits also detect ET-2 and ET-3. Therefore, the concentration of big ET-1 in perfusates was measured using the rat big ET-1 ELISA kit. Tissue processing At designated time points, animals were purchase Danoprevir perfused transcardially with phosphate-buffered saline followed by 23584186 2 Endothelin-1 in Seizure-Induced Vasogenic Edema 4% paraformaldehyde in 0.1 M phosphate buffer under urethane anesthesia. The brains were removed and postfixed in the same fixative for 4 hr. The brains were infiltrated with 30% sucrose overnight, frozen and sectioned with a cryostat at 30 m and consecutive sections were contained in six-well plates containing PBS. Every sixth section in the series throughout the entire PC was used for stereological study. For western blot, tissues were homogenized in 50 mM Tris containing 50 mM HEPES, ethylene glycol tetraacetic acid, 0.2% Tergitol type NP-40, 10 mM ethylenediaminetetraacetic acid, 15 mM sodium pyrophosphate, 100 mM glycerophosphate, 50 mM NaF, 150 mM NaCl, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol. After centrifugation, the protein concentration in the supernatant was determined using a Micro BCA Protein Assay Kit with bovine serum albumin as