Animals
Eight-week-old C57BL/6J mice weighing 25 to 30 g were used in the experiments. Animals were housed in groups of five per cage in a 22°C ± 2°C and 45% ~ 65% relative humidity environment under a normal light cycle room (12-h light/12-h dark; 8:00 a.m. light on ~ 8:00 p.m. light off). All animals had free access to food and water. All procedures were in accordance with the Dalian Medical University Guidelines for the proper care and use of Laboratory Animals and were approved by the Laboratory Animal Care and Use Committee of Dalian Medical University.
LPS treatment
Lipopolysaccharide (LPS,
Escherichia coli, serotype O55:B5, Sigma-Aldrich, St. Louis, MO, USA) was used to induce an inflammatory response. LPS was injected intraperitoneally at a dose of 5 mg/kg dissolved in sterile, endotoxin-free 0.9% saline vehicle. Control injections were equivolume vehicle. The dosage of LPS was based on a previous study of LPS-induced neurotoxicity [
46,
47].
Tissue preparation
At the time points of 6 h, 24 h, 72 h, 7 days, and 10 days after LPS injection, mice were anesthetized with diethylether and perfused with 4% paraformaldehyde solution. The brains were removed and post-fixed and then cryoprotected in 20% sucrose solution and embedded in OCT compound (McCormick Scientific, St. Louis MO, USA); and serial 18 μm sagittal sections were made with a cryostat (Leica CM 3050S, Solms, Germany) and used for the immunohistochemical (IHC) and in situ hybridization (ISH) studies.
For ELISA, mice were perfused with ice-cold PBS, and then the brains were removed and homogenized on ice. The homogenates were centrifugated at 12,000 g for 5 min at 4°C. The supernate was stored at −80°C.
In situ hybridization
ISH was performed as described previously [
48]. After hybridization of Cat H (NM_007801, 35 to 1,200 bp) cRNA probe, samples were incubated overnight with alkaline phosphatase-conjugated anti-digoxigenin (DIG) antibody (Roche, Basel, Switzerland) at 4°C. Color development was achieved by incubation with NBT/BCIP for 16 h at room temperature. Some sections were counterstained with Nuclear Fast Red for observation and analysis; others were processed for Iba-1, GFAP, and NeuN IHC staining after ISH, respectively. Images were captured using the Nikon digital camera system (DS-Fi1) in combination with microscopy (Nikon Eclipse 80i, Nikon, Tokyo, Japan). The number of cells expressing Cat H mRNA was counted with ImageJ 1.41 (National Institutes of Health, Bethesda, Maryland). Three sections from each mouse (with five mice per condition) were used for analysis.
Immunohistochemical staining
IHC was performed as described by Ma
et al. [
49]. The following antibodies were used: mouse anti-NeuN monoclonal antibody (1:1,000, Chemicon, EMD Millipore, Billerica, MA, USA), rabbit anti-GFAP polyclonal antibody (1:1,000, Dako, Glostrup, Denmark), rabbit anti-Iba1 polyclonal antibody (1:500, Wako, Osaka, Japan). Secondary antibodies were labeled with biotin (1:200, Vector Laboratory, Burlingame, CA, USA). After IHC reaction, images were captured using the Nikon digital camera system (DS-Fi1) in combination with microscopy (Nikon Eclipse 80i).
Real-time quantitative PCR
Total RNA was extracted from the brain using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Reverse transcription was performed using SuperScript II Reverse Transcriptase (Invitrogen). Real-time quantitative PCR was performed in 25 μL reaction volume using SYBR green PCR Master Mix (Thermo Scientific, Waltham, MA, USA), as described by the manufacturer. The primer sequences are the following: Cat H: forward: 5′ GAGCAGCAGCTGGTGGATTG 3′, reverse: 5′ CCATGATGCCCTTGTTGTATAGGA 3′; β-actin: forward: 5′ ATCATGTTTGAGACCTTCAACA 3′, reverse: 5′ CATCTCCTGCTCGAAGTCTA 3′. All primers were synthesized by Takara Biotechnology Co. Ltd. (Dalian, China). The thermal cycling conditions included denaturation step at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s and the final melting curve program with ramping rate of 0.5°C/s from 55°C to 95°C. Amplification specificity of PCR products was confirmed by melting curve analysis and agarose gel electrophoresis. Fold regulation values were calculated relative to the expression mean of the group displaying the lowest expression.
Cell culture
Primary microglia were harvested from primary mixed glial cell cultures prepared from neonatal C57BL/6J mice pups as previously reported (Fan
et al., [
18]). In brief, after the meninges were carefully removed, the neonatal brain was dissociated by pipetting. The cell suspension was plated in 10-cm culture dish at a density of one brain per two dishes in 10 mL DMEM (Sigma-Aldrich) containing 10% fetal bovine serum (FBS) (ICN Biomedicals, Santa Ana, CA, USA). After 14 to 21 days
in vitro, mixed glia cultures were dissociated by trypsinization, and the cell suspension was seeded on a petri dish and incubated for 30 min in a CO
2 incubator. Adherent cells were harvested as primary microglia. Microglia were reseeded in culture plates. The purity of microglia was approximately 99% as determined by CD11b (rat monoclonal IgG, clone M1/70, Abcam, Cambridge, UK) staining.
The immortalized murine microglial cell line BV2 was a kind gift from Dr. XF Wu (Dalian Medical University, China) and were maintained in DMEM supplemented with 10% FBS, 2 mM glutamine, and 100 U/mL penicillin/streptomycin at 37°C in 5% CO2 in a humidified atmosphere.
Prior to inflammatory treatments, primary microglia and BV2 cells were cultured in media without FBS for 24 h at 1.0 × 105 cells/24-well plate.
Cell treatment
BV2 cells were incubated for 24 h with LPS (10 ng/mL), then the culture media were collected, centrifuged to remove any cellular material.
Cat H antibody (N-18, Santa Cruz Biotech, Dallas, TX, USA) was added to the Cat H solution or microglial conditioned media and incubated for 1 h to allow sufficient time for antibody binding, then the samples were transferred to BV2 cells and incubated for 24 h. Controls were carried out in which Cat H antibody was directly added to BV2 cells to investigate non-specific effects.
Measurement of nitrite content
The production of nitric oxide (NO) in vitro was evaluated indirectly by measuring nitrite concentration (KeyGEN biotechnology, Nanjing, China). Test samples were the media obtained from BV2 cells and primary microglia following treatment with LPS (10 ng/mL), TNF-α (1 ng/mL), IL-1β (1 ng/mL), IL-6 (1 ng/mL), or IFN-γ (50 ng/mL) for 24 h, respectively. The absorbance at the wavelength 540 nm was determined using a microplate reader (iMark, Bio-rad, Hercules, CA, Japan).
ELISA
Cat H and proinflammation factors levels in the conditioned media were determined by enzyme-linked immunosorbent assay (ELISA).
Test samples for Cat H include brain supernate obtained from LPS-injected mice, the media from primary microglia and BV2 cells activated by LPS (10, 100, 1,000 ng/mL), IL-1β, IL-6, TNF-α (1, 10, and 100 ng/mL in each case), IFN-γ (50,500,5,000 ng/mL) for 24 h, respectively, and the media from BV2 cells treated with Cat H (2 ng/μL) in combination with Cat H antibody. Test samples for proinflammation factors were media from BV2 cells exposed to recombinant active Cat H protein (Abcam) in 0.2, 2, and 20 ng/μL, respectively.
The assay was performed according to Cat H ELISA Kit protocols (R&D) and TNF-α, IL-1β, IL-6, and IFN-γ ELISA Kit protocols (Peprotech, Rehovot, Israel). A microplate reader (iMark, Bio-rad, Japan) was used to measure absorbance at 450 nm. Cat H concentration was expressed in U/g of total protein and proinflammation factors in pg/μg of total protein.
Cat H enzymatic activity assay
Cat H aminopeptidase activity was determined by degradation of synthetic fluorogenic substrate L-Arg-7-amido-4-methylcoumarin (L-Arg-AMC). Test samples are the conditioned media obtained from primary microglia and BV2 cells following treatments with LPS (10 ng/mL), IL-1β, IL-6, TNF-α (10 ng/mL in each case), and IFN-γ (500 ng/mL) for 24 h, respectively. The procedures were performed according to Cat H assay kit protocols (Genmed Scientifics Inc., Wilmington, DE, USA). All samples including control groups (50 μg Cat H protein/well in 96-well plate) were incubated with the L-Arg-AMC at 37°C for 60 min. The release of fluorescent AMC was measured at an excitation and emission wavelength of 360 and 460 nm, respectively, in EnSpire Multimode Plate Reader (PerkinElmer, Waltham, MA, USA). The fluorescent signal was calibrated using known concentrations of AMC. Data were presented as relative folds to untreated control.
Flow cytometry
Flow cytometry was used to evaluate neuronal death following addition of recombinant active Cat H protein (Abcam) in 2 and 20 ng/μL to the culture media of the neuroblastoma cell line Neuro2a cells, respectively. After incubation for 24 h, the cells were harvested by centrifugation at 2,000 rpm for 5 min. After washing twice with pre-cold phosphate-buffered saline (PBS, pH 7.4), cells were resuspended in binding buffer and then stained by Annexin V-FITC and propidium iodine (PI) for 5 to 15 min at room temperature. At last, these cells were subjected to flow cytometer (Becton Dickinson, Laguna Hills, CA, USA).
In situ cell death detection
Adherent Neuro2a cells on coverslips were incubated with recombinant active Cat H protein (Abcam) in 20 ng/μL for 24 h. In situ cell death detection kit (TMR red, Roche) was applied to perform TdT-mediated dUTP nick end labeling (TUNEL) staining to evaluate neuronal apoptosis. Briefly, the cells were fixed by 4% paraformaldehyde in PBS (pH 7.4) for 1 h at 4°C, washed in PBS, and then incubated in permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min at 4°C. Later, the cells were incubated in TUNEL reaction mixture for 1 h at 37°C in the dark. Hoechst 33342 was used for the nuclear counterstaining. Negative control cells were used as label solution instead of TUNEL reaction mixture. Positive control cells were incubated with DNase I recombinant (Takara) for 10 min at room temperature to induce DNA strand breaks, prior to labeling procedures. The numbers of apoptotic cells were counted using an Olympus IX70 inverted fluorescence microscope. Cells were scored in eight randomly chosen fields under a magnification of × 400 per coverslip on at least two coverslips per treatment. The percentage of cells positive for apoptosis was calculated.
Statistical analysis
Data were expressed as mean + standard error of the mean (SEM) from three independent experiments. All statistical analyses were performed using the Statistical Package for Social Sciences (Version 17.0). One-way ANOVA test was used to detect significance of the differences among more than two arithmetic means, followed by post hoc Scheffe test to detect the differences between two means. Student’s t test was used to detect the significance of differences between two means. P < 0.05 was considered statistically significant.