The clinical and the animal experimental studies were approved by the Medical Ethics Committee of the Third Affiliated Hospital of Sun Yat-Sen University and the Animal Care and Use Committee of Sun Yat-Sen University respectively. All participants had signed the informed consent according to the principles illustrated in Declaration of Helsinki.
In this study, a total of 38 stroke patients recruited in the Third Affiliated Hospital of Sun Yat-Sen University from July 2018 to October 2019 consecutively had an independently documented primary stroke event in combination with confirmed magnetic resonance imaging (MRI) evidence showing ischemic stroke. The inclusion and exclusion criteria have been published previously (19). Clinical data, including age, gender, and score of National Institute stroke scale (NIHSS), were recorded. We estimated dietary sodium intake by measurements of 24-hour urinary excretion of sodium. Patient demographics including co-morbidities were summarized in Supplementary Table 1.
MRI Scanning and Infarct Volume Analysis of Patients
Magnetic resonance imaging (MRI) was performed within 24h of admission using 1.5- or 3.0-T magnetic resonance imaging (Signa; GE Medical Systems, Milwaukee, WI, USA). In this study, the diffusion-weighted imaging (DWI) spin-echo planar sequence included 20 contiguous axial oblique slices (b = 0 and 1000s/mm2 iso-tropically weighted; repetition time/echo time, 6000/60.4ms; acquisition matrix, 128 × 128; slice thickness, 5mm; interslice gap, 1mm; field of view, 24cm). DWI lesions in 38 patients were measured with Analyze 7.0 software (Analyze Direct, KS). Cerebral infarct sizes were assessed by largest infarct diameter determined on the image demonstrating the largest lesion (20-22). MRI scans of patients were assessed by experienced neurologist Zhengqi Lu, who was blinded to the patients’ clinical features. All images were interpreted with the same window settings, same types of monitors and lighting conditions.
Anti-coagulated blood (3mL) was collected, and then diluted 2-fold with PBS, pipetted into centrifuge tube prefilled with Ficoll lymphocyte separation solution (TBDscience), followed by centrifuged at 2000rpm for 25 minutes at room temperature without deceleration. PBMCs from the buffy coat were washed twice with PBS, then stored at –80°C until further analysis.
C57/Bl6 wild-type mice (8 weeks old, weight 18–25 g) were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China) and housed in a humidity- and temperature-controlled animal facility in Sun Yat-sen University with a 12-h light–dark cycle. Mice received normal chow (0.5% NaCl) and tap water ad libitum (normal diet) or sodium-rich chow (8% NaCl) and tap water containing 1% NaCl ad libitum (HSD) for 4 weeks according to the experiment.
Model of acute ischemic stroke
Mice were subjected to focal acute ischemic stroke induced with transient middle cerebral artery occlusion (tMCAO). Procedures of tMCAO were described previously (19). Briefly, mice were anesthetized with 1.5-2.0% isoflurane under conditions of spontaneous breathing. A filament was inserted into the external carotid artery (ECA) and was directed to the middle cerebral artery (MCA) through the internal carotid artery (ICA). Filament insertion into the ICA was maintained for 60min followed by reperfusion with maintenance of core body temperatures. Cerebral blood flow (CBF) during surgery was measured by laser Doppler flow cytometry. Mice with more than 70% reduction of blood flow in the ischemic core were included in the study and mice that died during surgery were excluded. Survival of mice were recorded.
Infarct volume analysis
For immunologic staining of NeuN, six equally spaced coronal brain sections encompassing the MCA territory were stained with NeuN antibodies. Infarct volume was analyzed with NIH Image J software on NeuN-stained sections. The infarct area was determined as the difference between the NeuN positive area of contralateral hemisphere and ipsilateral hemisphere. Brain infarct was determined by multiplying the mean area of tissue loss by the distances between the two adjacent stained brain slices.
Primary macrophage enriched culture and Stimulation
Primary macrophage-enriched cultures were prepared form the bone marrow of 6-8-week-old healthy C57/Bl6 wild-type mice using EasySep Mouse Monocyte Enrichment Kit (Stem cell) according to manufacturer’s instructions. Macrophages were induced with MCSF (50ng/ml) for 6d in macrophage culture medium (RPMI1640 + 10%FBS). For polarization, macrophages were treated with lipopolysaccharide (LPS, 100ng/mL, Sigma), or IL-4 (20ng/mL, Peprotech) for 24 hours.
Primary microglia culture
Primary mouse microglia were obtained from BLUEFBIO company, and was cultured in culture medium (DMEM-HG + 10%FBS) until treatment.
Primary cortical neurons culture and OGD
Primary cortical neuronal cultures were prepared from E16–18 embryos of C57/Bl6 mice as previously described (23).
Neuronal ischemia was induced with OGD. Briefly, culture medium (Neural basal + B27 + 2% glutamate) was retreated
and was replaced by EBSS (Gibco). Neurons were then incubated in 95% N2 + 5% CO2 for 90 min.
For evaluation of efferocytic capacity, apoptotic neurons were labelled with the dead cell marker Propidium iodide (PI) in PBS (1ug/ml, 37℃, 15min) and treated to macrophages, with a ratio of dead neurons : macrophages = 5:1, for indicated time periods. For in vitro immunol staining experiments, macrophages were pre-grown on poly-l-lysine coated cover slips. The cover slips of macrophage were washed for 2 times to remove unengulfed neurons and fixed with 4% paraformaldehyde. The cover slips were then subjected to immunol staining and removed from wells using tweezers and mounted to the slides. F-actin of macrophage was then stained with Alexa Fluor488 phalloidin (A12379, 1:500 in PBS; Invitrogen) at room temperature in the dark for 30min. For flow cytometry experiment, macrophages were pre-seeded on 24-well plates and treated with the same ratio of dead neurons for indicated time periods. Macrophages were washed with PBS and detached from wells with trypsin and were subjected to flow cytometric analysis. Percentage of efferocytic macrophages (PI+) was calculated with flow cytometric analysis.
Lentiviral infection of macrophage
Lenti virus was constructed and packaged by FenghBio (Changsha, China). The macrophage culture was infected for 3d with Lenti-TREM2 or the control vectors. The overexpression of TREM2 was confirmed by western blot and flow cytometry.
Flow cytometric analysis
Brain tissue was homogenized and prepared as single-cell suspensions for flow cytometric analysis (FACS). Briefly, brains were dissected, and ipsilateral hemispheres were collected. Each hemisphere was subjected to digestion with 0.25% trypsin-EDTA (Thermo Fisher, Carlsbad, CA, USA) at 37 °C for 25 min. Brain tissue was then pressed through a cell strainer (70 μm). Brain cells were separated from myelin debris by centrifugation in 30%/70% Percoll solution (GE Healthcare Biosciences AB, Uppsala, Sweden). Brain cells at the interface were collected, washed with HBSS, and subjected to further staining. The following antibodies were used: CD45-PE-Texas Red (1:400, Biolegend), CD11b-PE (1:400, Biolegend), CD3-PerCp/Cy5.5 (1:400, Biolegend), CD19-FITC (1:400, Biolegend), Ly6G-APC/Cy7 (1:400, Biolegend), TREM2-PE (1:200, R&D Systems), TNFα-PE (1:200, Biolegend), CD206-Alexa Fluor 647 (1:200, BD bioscience), Arg1-APC (1:200, R&D Systems). FACS was performed using a fluorescence-activated cell sorter flow cytometer (BD bioscience, San Diego, CA), and data were analyzed using FlowJo X 10.0.7r2 software. Appropriated isotype controls were stained following the manufacturer’s instruction (Thermo Fisher, Carlsbad, CA, USA). Fluorchrome compensation was performed with single-stained OneComp eBeads (Thermo Fisher, Carlsbad, CA, USA). As for data presentation, when cells could be divided into negative or positive populations, percentage of cells was calculated. When expression of coordinated marker was consecutive and population separation was obscure, data were presented as mean fluorescence intensity (MFI).
Immunofluorescence staining and cell quantification
Animals were euthanized and perfused with PBS followed by 4% paraformaldehyde. After sufficient perfusion, brains were removed and then cut into 25μm frozen cryo-sections using a microtome. Brain sections and were incubated with primary antibodies at 4°C overnight. After washing with PBS, sections were incubated with secondary antibodies for 1h at room temperature. Sections were then washed and mounted with DAPI Fluoromount-G (Thermo Fisher, Carlsbad, CA, USA). The following primary antibodies were used: rabbit anti-NeuN (1:500 Abcam), rabbit anti-Iba1 (1:1000, Wako Pure Chemical Industries), goat anti-CD206 (1:500, R&D Systems), and rat anti-CD16 (1:500, Santa Cruz Biotechnology). The following secondary antibodies were applied: anti-rabbit secondary antibody conjugated with Cy3 (1:1000, Jackson ImmunoResearch Laboratories), anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (1:1000, Jackson ImmunoResearch Laboratories), anti-goat secondary antibody conjugated with Alexa Fluor 488 (1:1000, Jackson ImmunoResearch Laboratories), and anti-rat secondary antibody conjugated with Alexa Fluor 488 (1:1000, Jackson ImmunoResearch Laboratories). For neuronal apoptosis analysis, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was processed after NeuN labeling according to instructions from the manufacturer (Thermo Fisher). Confocal microscopy images were acquired using a Leica SP confocal microscope and Leica confocal software. Immunopositive cell quantification and area analysis were performed with the software of ImageJ (National Institutes of Health) by an investigator who was blinded to the experimental design. In quantification of cell in stroke penumbra, the stroke core was identified as the region in which the majority of DAPI-stained nuclei were shrunken, and the stroke penumbra was defined as the region of generally morphologically normal cells, approximately 450–500μm wide, surrounding the stroke core.
Quantitative determination of mRNA expression
Total RNA from cells was extracted with commercial kit (ESscience) according to the manufacturer’s instructions. A total of 1ug RNA (OD260nm/280nm = 1.8-2.2) was applied to the first strand cDNA synthesis in a 40ul system using PrimeScript RT reagent kit (Takara). Real time polymerase chain reaction (RT-PCR) was performed on a QuantStudio 5 (ABI) quantitative PCR machine using TB green Premix Ex Taq kit (Takara) with 1ul of the synthesized cDNA in each reaction with addition of ROX. The following program was performed: 95℃ for 30s; 95℃ for 5s and 60℃ for 34s, repeated for 40 cycles; 95℃ for 15s, 60℃ for 1min and 95℃ for 15s (Melt curve). Primers used in the study are listed in Supplementary Table 2. Double delta CT were calculated, and the data presented as fold change normalized to PBS-treated contralateral brain, PBS-treated macrophage, or negative control lentivirus-treated macrophage. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a normalizer housekeeping gene. In data analysis in Figure 2, Figure 5, and Supplementary Figure 1B, the mRNA expression level was visualized with heat map and clustered with the software of R using the “pheatmap” package.
Protein isolation was performed as previously described (24). Western blots were performed using the standard SDS-polyacrylamide gel electrophoresis method and enhanced chemiluminescence detection reagents (GE Healthcare Biosciences AB, Uppsala, Sweden). Antibodies against TREM1 (1:1000, Abcam), TREM2 (1:1000, Abcam), TNFα (1:1000, Proteintech), IL-10 (1:1000, Proteintech), β-actin (1:3000, Abcam), and GAPDH (1:3000, Cell signaling technology) were used according to the manufacturer’s directions. Immunoreactivity was semi-quantitatively measured by gel densitometric scanning and analyzed by the MCID image analysis system (Imaging Research, Inc.).
All results were presented as mean ± standard error of the mean (SEM). The differences in the means among multiple groups were analyzed using one- or two-way analysis of variance (ANOVA). When ANOVA showed significant differences, pair-wise comparisons between means were tested by Dunnett’s test. The Student’s t test was used for two-group comparisons. The software used for statistical analysis was R v3.6.3. In all analysis, P < 0.05 was considered statistically significant.