Chemicals and compounds
Methylscopolamine (Cat. # S2250), terbutaline (Cat. #T2528), and pilocarpine (Cat. #P6503) were purchased from Sigma-Aldrich. Parenteral diazepam (Hospira, Lot #30561) was purchased from Henry Schein. Indomethacin was purchased from Tocris Bioscience (Cat. #1708) and celecoxib was purchased from Cayman Chemical (Cat. # 10008672). Fluoro-Jade B (Cat. #AG310) was purchased from Sigma-Aldrich. Compound PBCH was kindly provided by Dr. Jae Yeol Lee [17, 18] and authenticated using LC/MS and NMR in the Medicinal Chemistry Core at the University of Tennessee Health Science Center.
Young adult male C57BL/6 mice (~ 8 weeks old) were purchased from Charles River Laboratories, housed under a 12-h light/ dark cycle in standard relative humidity at room temperature, and provided free access to food and water. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Tennessee Health Science Center and carried out carefully in full compliance with the Guide for the Care and Use of Laboratory Animals (Eighth Edition) provided by the National Institutes of Health (NIH).
Pilocarpine model of SE and drug treatment
Mice were pre-treated with methylscopolamine and terbutaline (2 mg/kg each in saline, i.p.) to minimize the unwanted effects of pilocarpine in peripheral organs. Thirty minutes later, pilocarpine (280 mg/kg in saline, freshly prepared, i.p.) was administered to induce seizures in mice. Seizures were classified using a modified Racine scale as we previously described [19, 20]. 0: normal behavior—grooming, walking, exploring, and sniffing; 1: immobile, staring, jumpy, and curled-up posture; 2: automatism—repetitive blinking, head bobbing, chewing, vibrissae twitching, face-washing, scratching, and “star-gazing”; 3: partial body clonus, sporadic myoclonic jerks, and shivering; 4: full body clonus, “corkscrew” turning and flipping, loss of posture, rearing, and falling; 5: onset of SE—nonintermittent seizure activities; 6: bouncing, wild running, and tonic seizures; 7: death. SE was defined by nonintermittent seizure activities and usually indicated by continual generalized clonic seizures without returning back to low-stage seizures. SE proceeded for 1 h and was terminated by diazepam (10 mg/kg, i.p.). Two hours after SE onset, that is, 1 h after administration of diazepam, animals were randomized for treatment with either compound PBCH (10 mg/kg, i.p.) or vehicle (10% DMSO, 50% PEG 400, 40% ddH2O). Mice were treated again at 8 and 20 h after SE onset. During recovery from SE, mice were fed moistened rodent chow, monitored daily, and given 5% dextrose in lactated Ringer’s solution (Baxter) when needed. Four days after SE, animals were euthanized under deep anesthesia with isoflurane and perfused with cold PBS to wash blood out of the brain. The hippocampal and cortical tissues were then collected for biochemical and histological analyses.
PGE2 in the brain tissues was measured using the PGE2 Multi-Format ELISA Kit (Arbor Assays, Cat. # K051). A total of 50 µL diluted lysate of each cortical sample was used for PGE2 measurement following the manufacturer’s protocol as we previously described [9, 21]. The absorbance was measured using a Synergy H1 microplate reader (BioTek) at 450 nm. A standard curve for PGE2 was run with each experiment. The PGE2 levels were normalized to tissue weights for comparisons between experimental groups.
COX inhibition assay
The COX Colorimetric Inhibitor Screening Assay Kit (Cayman Chemical, Cat. #701050) was used to evaluate the inhibition percentage of COX enzyme by tested compounds following the manufacturer’s manual. Non-selective COX inhibitor indomethacin and selective COX-2 inhibitor celecoxib were used as control compounds. A plate reader (Synergy H1) was used to measure the absorbance at 590 nm.
The mRNA expression levels of examined genes were quantified by quantitative PCR (qPCR) as we described previously [9, 22]. The total RNA from mouse brain tissues was isolated using TRIzol and the PureLink RNA Mini Kit (Invitrogen). RNA purity and concentration were measured using A260/A280 ratio and A260 value measured by a microvolume spectrophotometer (NanoDrop One, Thermo Fisher). The complementary DNA (cDNA) was synthesized using the SuperScript III First-Strand Synthesis SuperMix (Invitrogen). The qPCR was performed using cDNA, primers, and 2 × SYBR Green SuperMix with a final reaction volume of 20 µL in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories). Cycling conditions were set as: 95 °C for 2 min followed by 40 cycles of 95 °C for 15 s and then 60 °C for 1 min. Melting curve analysis was utilized to validate the specificity of the PCR products. Fluorescent data were obtained at the 60 °C step. The cycle of quantification for GAPDH was subtracted from the cycle of quantification measured for each gene of interest to yield ∆Cq. Samples that did not undergo cDNA synthesis served as negative controls. The sequences of primers used for qPCR are as follows: CCL2, forward 5’-CATCCACGTGTTGGCTCA-3’ and reverse 5’-GCTGCTGGTGATCCTCTTGTA-3’; CCL3, forward 5’-TGCCCTTGCTGTTCTTCTCT-3’ and reverse 5’-GTGGAATCTTCCGGCTGTAG-3’; CCL4, forward 5’-CATGAAGCTCTGCGTGTCTG-3’ and reverse 5’-GGAGGGTCAGAGCCCATT’; GAPDH, forward 5’-TGTCCGTCGTGGATCTGAC-3’ and reverse 5’-CCTGCTTCACCACCTTCTTG-3’; GFAP, forward 5’-GACAACTTTGCACAGGACCTC-3’ and reverse 5’-ATACGCAGCCAGGTTGTTCT-3’; Iba1, forward 5’-GGATTTGCAGGGAGGAAAAG-3’ and reverse 5’-TGGGATCATCGAGGAATTG-3’; IL-1β, forward 5’-TGAGCACCTTCTTTTCCTTCA-3’ and reverse 5’-TTGTCTAATGGGAACGTCACAC-3’; IL-6, forward 5’-TCTAATTCATATCTTCAACCAAGAGG-3’ and reverse 5’-TGGTCCTTAGCCACTCCTTC-3’; TNF-α, forward 5’-TCTTCTGTCTACTGAACTTCGG-3’ and reverse 5’-AAGATGATCTGAGTGTGAGGG-3’.
Coronal brain sections (25 µm) were fixed by 4% paraformaldehyde and underwent 10-min permeabilization by 0.2% Triton X-100, followed by blocking with 10% goat serum in PBS for 60 min. The sections were then incubated in rabbit anti-Iba1 polyclonal antibody (Wako Chemicals, Cat. # 019-19741, 1:200) or rabbit anti-GFAP polyclonal antibody (Thermo Fisher, Cat. # PA1-10019, 1:500) at 4 °C overnight. Sections were then washed and incubated with anti-rabbit secondary antibody conjugated with Alexa Fluor 546 (Invitrogen, Cat. # A-11035, 1:1000) for 1 h at room temperature. After washing, sections were stained with DAPI for 10 min and carefully mounted onto slides using ProLong Gold antifade mountant (Invitrogen, Cat. # P36930). Digital images were captured using a BZ-X800 fluorescence microscope (Keyence), and the image processing and quantitative analyses were performed using the ImageJ/Fiji software (NIH).
Fluoro-Jade B staining
Fluoro-Jade B (FJB) reagent is a neuron-specific anion which can selectively label degenerating neurons , and was used to detect SE-induced neuronal death in this study as we previously described [24,25,26]. In brief, coronal brain sections were successively immersed in 80% alcohol containing 1% NaOH for 5 min, in 70% alcohol for 2 min, and in distilled water for 2 min. Sections were then incubated in 0.06% potassium permanganate for 30 min with gentle agitation. After rinsing in distilled water for 1 min, sections were transferred to the FJB solution (0.0004, w/v, in distilled water with 0.1% acetic acid) for 30 min with gentle agitation in the dark. Sections were rinsed with three 1-min changes of distilled water, rapidly dried, and covered by the coverslip with DPX mountant. Images were captured by a BZ-X800 fluorescence microscope (Keyence), and the FJB-positive cells were counted in the sections between bregma − 1.5 and − 3.
Statistical analyses were performed using GraphPad Prism software by t test or Mann–Whitney U test as indicated in each experiment. P < 0.05 was considered statistically significant. All data are presented as mean + or ± SEM.