BV2 microglial cell line

BV2 cell line is the most frequently used substitute for primary microglia and has been used in studies related to neurodegenerative disorders ﻿[23]. In the present study, BV2 cells were cultured in DMEM (Corning, Manassas, VA, USA), supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% antibiotics (penicillin 100 U/mL, streptomycin 100 μg/mL), and were kept in an incubator at 37 °C, 5% CO 2 , and 95% relative humidity.

Animals

B6.129P-Cx3cr1tm1Litt/J mice (The Jackson laboratory) possess microglia with a fluorescent protein, which expresses fluorescence when the microglia are activated by stimuli such as inflammation and damage. The mice were raised in the National Laboratory Animal Center (NLAC) in Taiwan and housed and maintained on a 12-h-on/12-h-off light/dark cycle. All of the animals were allowed free access to food and water. The maintenance of the mice and the experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals [24] and the study was approved by the animal ethical committee of Medical College of National Taiwan University.

Chemicals and drugs

Lipopolysaccharide (LPS), KA, minocycline, everolimus, and rapamycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The primary antibodies Akt and GAPDH used for Western blotting were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and Genetex (Irvine, CA, USA), respectively. The other primary antibodies, including ERK and phosphor-ERK, used for Western blotting were purchased from Cell Signaling (Danvers, MA, USA).

MTT assay for cell viability

Before the nitrite assay and the qPCR assay, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to assess whether the drugs and the combination of drugs at the specific concentrations we used affect the cell viability. BV2 cells at a concentration of 1.5 × 105 cells/well were seeded into 24-well plates overnight. After treatment with different drugs for 24 h, MTT (Sigma-Aldrich, St. Louis, MO, USA) was added to each well at the final concentration of 0.5 mg/mL. After 3 h of incubation, the medium was removed and 500 μL of DMSO was added to each well. After 15 min of shaking for thorough mixing of DMSO and formazan, 200 μL of the mixture from each well was collected and placed into 96-well plates. The optical density was measured at 570 nm using a spectrophotometer. The amount of formazan formed directly correlates well with the number of live cells in the culture.

Nitrite assay

Nitrite is a metabolite of NO, and NO production can be measured through quantification analysis of nitrite production [25]. BV2 cells at a concentration of 1.5 × 105 cells/well were seeded into 24-well plates overnight. After treatment with different drugs for 24 h, 100 μL of medium from each well was collected and placed into 96-well plates, mixed with 100 μL of Griess reagent (Sigma-Aldrich, St. Louis, MO, USA) and shaken for 15 min to measure the nitrite amount. The optical density was measured at 562 nm using a spectrophotometer. The amount of nitrite in medium correlates well with the NO production by the cells.

Real-time PCR

BV2 cells at a concentration of 4.5 × 105 cells/3 mL were seeded into 40-mm dishes overnight. After treatment with different drugs for 24 h, total RNA was extracted by TRIZOL, and reverse transcribed to cDNA using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA). For real-time PCR, Maxima SYBR Green qPCR Master Mix (Thermo Scientific, Waltham, MA, USA) was used. For detecting iNOS, mTOR, NLRP3, and IL-1β level, we used the following primer sequences: iNOS, forward 5′-CTG CAT GGA ACA GTA TAA GGC AAA C-3′ and reverse 5′-CAG ACA GTT TCT GGT CGA TGT CAT GA-3′; mTOR, forward 5′-ACT GAG GAG GGA GAA CAG CA-3′ and reverse 5′-TGG CTC CAT CTG CTA GTG TG-3′; NLRP3, forward 5′-AGA GCC TAC AGT TGG GTG AAA TG-3′ and reverse 5′-CCA CGC CTA CCA GGA AAT CTC-3′; IL-1β, forward 5′-CCC TGC AGC TGG AGA GTG TGG A-3′ and reverse 5′-TGT GCT CTG CTT GTG AGG TGC TG-3′; and β-actin, forward 5′-CTA AGG CCA ACC GTG AAA AG-3′ and reverse 5′-ACC AGA GGC ATA CAG GGA CA-3′. Relative amounts of the indicated mRNA levels were determined by the 2−ΔΔCT method, normalizing with β-actin levels.

KA-induced seizures and the two-hit seizure model

The severity of seizures induced by KA can be distinguished using the modified Racine’s scale based on the abnormal behavior of mice as follows: stage I—chewing; stage II—head nodding; stage III—unilateral forelimb clonus; stage IV—bilateral forelimb clonus; stage V—bilateral forelimb clonus and falling; stage VI—running or bouncing seizure; stage VII—tonic hindlimb extension; and stage VIII—tonic hindlimb extension culminating in death [26–28]. The behavior from stage III to stage VIII can be recognized in the present mouse model. The rearing and falling behavior of stage V can be easily demonstrated in mice. Mice exhibiting at least stage V were included in this study due to microglial activation and neuronal loss in this stage [28–32]. The latency was recorded when mice first showed rearing behavior of stage V in this study. Koh et al. [33] developed a two-hit seizure model, demonstrating that “an early-life seizure permanently decreases seizure threshold and increases the susceptibility to seizure-induced cell death in adulthood”. In addition, they showed that anti-inflammatory therapy with minocycline after the initial status epilepticus blocked the epileptogenic process and mitigated the long-term damaging effects of early-life seizures [34].

Based on the 7-day protocol of Koh et al. with some modification, in this study, the postnatal day 25 (P25) mice received intra-peritoneal injection of KA 25 mg/kg on days 1 and 7. The durations from injection to stage V seizures on days 1 and 7, which were defined as latency 1 and 2, respectively, were recorded. Three hours after the seizure onset on day 1 and the following days until day 6, mice were injected with everolimus or PBS as control q.d. intraperitoneally. Mice were divided into the following three groups: KpK group, KA injection on days 1 and 7 and PBS from day 1 to day 6; KeK group, KA injection on days 1 and 7 and everolimus from day 1 to day 6; and PpP group as controls, PBS injection throughout the experiment. A 14-day protocol was followed with the same procedures: the first and second KA injection on days 1 and 14 and everolimus or PBS from day 1 to day 13.

Quantification of microglial activation

Five to six mice were analyzed per group. Mice were sacrificed and perfused with PBS and 4% paraformaldehyde/0.1 M sodium phosphate buffer. The brains were harvested and kept in 4% paraformaldehyde/0.1 M sodium phosphate buffer for post-fixation. Before slicing the brains, they were kept in 30% sucrose/4% paraformaldehyde solution. Then the brains were cut into 30-μm horizontal slices until the hippocampus was revealed. The slices were then collected every six slices, and at least six slices were gathered for each brain. Images of the hippocampus CA1 and CA3 regions were taken by a fluorescence microscope and a camera under ×10 objective. All images were captured under identical settings. The activated microglial cells exhibited fluorescence. The number of activated microglial cells in each slice was counted within the CA1 and CA3 regions in each animal. The mean number of activated microglial cells was calculated by the Image J software. Data were expressed as the mean of activated microglial cells in CA1 or CA3 regions per slice in each animal.

Western blotting

For in vitro experiment, the BV2 cells were seeded at a concentration of 1.5 × 106 cells/10 mL into 100-mm dishes overnight. After treatment with different drugs for 24 h, the cell lysates were collected for protein analysis. For in vivo experiment, 5 to 11 mice were sacrificed 24 h after KA injection on day 7, and the hippocampi were resected and stored in liquid nitrogen. After homogenization, protein expression was analyzed by Western blotting. After electrophoresis and transfer to nitrocellulose membranes, the membranes were blocked with 5% bovine serum albumin (BSA) in PBST (PBS and 0.05% tween-20) for 30 min, incubated with primary antibodies in 5% BSA solution overnight, and then incubated with secondary antibodies in 5% BSA solution for 1 h. Signals were visualized using the ECL reagent and a chemiluminescence and fluorescence image analyzer. ImageJ software was used to subtract background and to perform densitometry.

Statistical analysis

For in vitro and in vivo experiments, one-way ANOVA test was used to analyze cell viability, nitrite production, mRNA levels, and protein phosphorylation among the different treatment groups, with post hoc comparison by LSD test. Seizure severity at days 1 and 7 was compared using chi-square test. All data were analyzed using IBM® SPSS® Statistics software version 19.0 (IBM Inc., Somers, NY, USA). A p value <0.05 was considered to be statistically significant.