Primary microglial cell isolation from newborn rat brain
We obtained primary microglial cells, according to a previously described technique [28,29]. Briefly, mixed glial cultures were prepared from newborn to 3-day-old Sprague-Dawley rat brains containing the cortex, hippocampus and striatum (CLEA Japan Inc., Tokyo, Japan) and cultured until confluent as described. The cells were cultured in high glucose Dulbecco’s modified Eagle medium (DMEM high glucose, WAKO, Osaka, Japan) containing 10% fetal bovine serum (FBS) and penicillin/streptomycin at 37°C in a 10% CO2 incubator. After approximately 2 weeks, microglial cells were harvested by the shaking method and replated on 6-well plates at 2×106 cells/well, 96-well plates at 1×105 cells/well or 8-well chamber slides at 2×105 cells/well. The microglial cell cultures consisted of >98% microglial cells (stained with anti-Iba1 antibody (WAKO)) (Supplementary Fig. 1). All animal experiments were performed with the permission of the Animal Experiment Ethics Committee of the National Center of Neurology and Psychiatry.
Experimental groups and oxygen glucose deprivation and LOX-1 knockdown treatments
We performed oxygen glucose deprivation (OGD) treatment as an in vitro model of hypoxia/ischemia [30,31]. The harvested microglial cells were cultured in high glucose DMEM containing 10% FBS for 24 hours. Then, the culture medium was replaced with glucose-free DMEM without FBS. Primary microglial cells were treated with OGD and LOX-1 knockdown.
For OGD treatment, cultured microglial cells were treated with a BIONIX-2 hypoxic cell culture kit (Sugiyamagen, Tokyo, Japan). The concentration of oxygen was maintained at almost 0%. For the controls, the culture medium was replaced with high glucose DMEM without FBS and microglial cells were cultured under normal oxygen concentrations. After OGD treatment for 6 hours, the microglial cells were washed with PBS. Then, total RNA or proteins were extracted for analysis and the culture supernatant was collected for cytokine measurement. For immunocytochemistry, the cells in chamber slides were washed with PBS and then fixed with 4% paraformaldehyde (PFA).
For LOX-1 knockdown, the expression of LOX-1 in primary microglial cells was silenced using the small interfering RNA (siRNA) Silencer Select rat Olr1 (genetic name of LOX-1) (Thermo Fisher Scientific, Waltham, MA) with Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific), according to the manufacture’s instructions. We used Silencer Select negative control No.1 siRNA that did not target any rat genes as a negative control. The diluted siRNA (12.5 pmol/ml) and transfection reagent (3.75 μl/ml) were added to the microglial cell culture. The cells were incubated with the siRNA for 24 hours and then were subjected to the OGD experiments.
Inhibitors of p38-MAPK and NF-kB
SB203580 (199-16551, Wako Pure Chemical Industries, Osaka, Japan), a p38-MAPK inhibitor, was added to cultured microglial cells at a concentration of 20 μmol/l for 60 minutes before OGD treatment . BAY11-7082 (19542-67-7, Wako Pure Chemical Industries), an NF-κB inhibitor, was added to the isolated microglial cells at a concentration of 10 μmol/l for 30 minutes before OGD treatment .
RNA extraction and real-time quantitative PCR
Total RNA was extracted from rat brains and cultured microglial cells using the RNeasy Plus Mini kit (Qiagen, Venlo, NLD), according to the manufacturer’s protocols. Then, the total RNA concentration was measured using a Nanodrop (Thermo Fisher Scientific). For RT-PCR, cDNA was prepared from total RNA using a high-capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific). RT-PCR was performed using LightCycler 480 SYBR Green I master mix (Roche, Basel, Switzerland) on a LightCycler 480 System II (Roche, Basel, CHE). The experiments were performed in triplicate. The mRNA levels were normalized relative to the endogenous reference gene act-b (β-actin). The results are described as the fold change of the Ct value relative to the control groups.
Whole transcriptome microarray analysis was performed using a Rat Clariom S assay (Thermo Fisher Scientific) according to the manufacturer’s instructions. Analysis and normalization of the raw data was conducted using Transcriptome Analysis Console (TAC) 4.0 (Thermo Fisher Scientific). Differentially expressed genes (DEGs) were determined as genes with an FDR < 0.01 and more than 2-fold change. Gene set analysis of gene ontology (GO) biological processes and KEGG pathways was conducted using GSEA software (http://software.broadinstitute.org/gsea/index.jsp).
Protein extraction and immunoblotting
Total proteins were extracted using RIPA buffer (Nacalai Tesque, Kyoto, Japan) according to the manufacturer’s instructions. Separate cytoplasmic and nuclear protein fractions were extracted using NE-PE nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific). Then, the protein concentrations were measured using a Pierce BCA protein assay kit (Thermo Fisher Scientific).
Twenty micrograms of the extracted proteins were loaded in each lane of TruPAG Precast Gels 4-12% (Sigma-Aldrich Corporate, St. Louis, MO) for electrophoresis. After transfer to a polyvinylidine difluoride membrane, each immunoreacted band was detected using Amersham ECL prime western blotting detection reagent (GE Healthcare, Boston, MA) according to the manufacturer’s instructions. As a reference, β-actin was detected using a specific antibody. The expression levels of the detected bands were measured and calculated by ImageQuant TL (GE Healthcare).
The microglial cells on chamber slides or in 96-well plates were washed with PBS and fixed with 4% PFA for 30 minutes. After that, the cells were incubated with the primary antibodies. Then, the cells were incubated with Alex Flour conjugated secondary antibodies (Thermo Fisher Scientific) for 1 hour and were mounted with Hoechst 33342 (Thermo Fisher Scientific). The fluorescent samples were observed with confocal laser scanning microscopy (LSM780; Zeiss, Oberkochen, Germany) or fluorescence microscopy (IX71; Olympus, Tokyo, Japan).
The microglial cell culture medium was collected and centrifuged for 20 minutes at 1,000xg. Then, cytokine/chemokine concentrations in the supernatants were measured using a BioPlex ProTM Rat Cytokine 23-Plex Assay (BioRad, Hercules, CA).
Reactive oxygen species (ROS) detection
After the OGD experiments, microglial cells were subjected to OGD treatment and treated with 4.5% glucose containing original medium as controls. Then, cellular ROS levels were detected using CellRox Green Reagent (C10444: Thermo Fisher Scientific). Briefly, the microglial cells were stained with 5 μM CellRox Green Reagent by adding the probe to the complete media and incubating at 37°C for 30 minutes. Fifty μM of N-acetyl cysteine (NAC), an antioxidant was added to some of the OGD-treated wells. Then, the cells were washed with PBS and were fixed with 4% PFA. Finally, the microglial nuclei were stained with Hoechst 33342 (Roche). The cells were observed with the microscope (IX71: Olympus, Tokyo, Japan) and the mean fluorescent intensities per cells were analyzed using In Cell Analyzer 2000 (GE Healthcare).
After the OGD experiments, the microglial cells conducted OGD treatment were added with 4.5% glucose as the same glucose concentrations as the CTL. Then, the cells were incubated for 2 hours with CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WA) at 37 °C according to the manufacturer’s instructions. This reagent contains a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS]. The quantity of formazan product as measured by absorbance at 490 nm is directly proportional to the number of living cells in cultures. After the 2 hours incubation, the absorbance at 490 nm was measured using a 96-well plate reader.
All data were described as means ± SD. Data were analyzed with one-way ANOVA followed by Tukey’s post hoc test or unpaired t-test. Statistical analysis was performed using IBM SPSS version 22 (IBM, Armonk, NY). P < 0.05 was considered to be statistically significant.