Animals
Eight-to-ten weeks old male C57BL/6 mice, weighing 21–25 g, were obtained from Daehan Biolink Co. Ltd. (Chungbuk, Korea) and randomly divided into animal cages. Animals were housed and maintained in a controlled environment at 22–24°C and 55% humidity with 12 h light/dark cycles and fed regular rodent chow and tap water ad libitum. All animal care was guided by the Seoul National University Institutional Animal Care and Use Committee (SNU IACUC) to minimize pain and distress during experimental interventions and all animal experimental procedures were performed following the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines.
Stereotaxic spike protein delivery to mice hippocampi
Following a previous study, hippocampal administration of S1 protein was performed27. Eight-week-old mice were used for the spike protein injections. S1 protein was purchased from Acrobiosystems (S1N-C52H4; Newark, DE, USA), and used after polymixin B (Sigma-Aldrich, St. Louis, MO, USA) treatment (30 µg/ml). The animals were anesthetized with isoflurane, and the injection paths were drilled into their skulls. Then, 5 µg of S1 protein (1 µg/µl) were bilaterally injected into each hippocampal region using a Hamilton syringe (Hamilton Company, Reno, NV, USA) attached to a syringe pump (Stoelting Co., Wood Dale, IL, USA) at a constant volume of 0.5 µl/min. The injection coordinates of the hippocampus were 1.5 (ML), −2.06 (AP), and −2.0 (DV) from the bregma.
Novel object recognition and location recognition test
The novel object recognition and location recognition tests were performed using previously described methods28, 29 with minor modifications. First, each mouse was placed on one side of the open field box and allowed to freely explore for 10 min. After one day, two identical objects were presented to the mouse for 10 min. Then, one object was replaced with a novel object, and the mouse was allowed to explore for 5 min. Subsequently, one object was replaced in a novel location, and the mouse was allowed to explore for 5 min. The chamber and objects were cleaned with 70% ethanol between trials to remove olfactory stimuli. Testing sessions were video monitored, and object exploration times were scored by a blinded experimenter. Results are expressed as the discrimination ratio of time spent with the novel object or novel location to the total exploration time. The discrimination index of the novel object or location was calculated by the following formula: (discrimination time of novel object or location/total discrimination time of novel and familiar object or location)*100. The exploration time were measured automatically by SMART 3.0 software (Panlab, Barcelona, Spain).
Elevated plus maze (EPM)
The EPM was used to examine anxiety-like behavior. The behavioral apparatus consisted of two open arms and two closed arms elevated 50 cm above the floor. Mice were placed individually in the center of the maze, facing an open arm, and allowed to freely explore for 5 min. The maze was cleaned with 70% ethanol after each test to prevent influence from the previously tested mouse. The times spent in the open and closed arms were measured automatically by SMART 3.0 software.
Open field test (OFT)
The OFT was performed as previously described30. Each mouse was placed in the center of an open arena and allowed to freely explore the arena for 5 min. The times spent in the center and the periphery zone were automatically measured by SMART 3.0 software.
Cresyl violet staining
Cresyl violet staining was performed as previously described with minor modification31. Briefly, brain sections were rehydrated with serially diluted ethanol (from 100 to 70%) for 2 min and with xylene for 5 min. Rehydrated tissues were incubated with 0.1% cresyl violet solution for 10 min. After the solution was washed away with distilled water, tissues were dehydrated with diluted ethanol (from 70 to 100%) for up to 1 min for each. The samples were rinsed in xylene and mounted on slides.
Immunohistochemistry Immunostaining
was carried out using previously established protocols32. Briefly, the brain sections were incubated in a blocking solution (5% normal goat serum, 2% bovine serum albumin, and 0.1% Triton X-100) for 1 h at room temperature (RT). Tissue samples were then incubated overnight at RT with primary antibody for rabbit-anti-Iba1 (1:1000; Wako, Osaka, Japan) and mouse-anti-GFAP (1:2000; Millipore, Darmstadt, Germany) or NeuN (1:1000; Millipore). After being rinsed in 0.1 M PBS, the samples were incubated for 1 h at RT with a mixture of Cy3- or FITC-conjugated secondary antibodies (1:200; Jackson ImmunoResearch, West Grove, PA, USA) and mounted with VectaShield medium (Vector Labs, Burlingame, CA, USA). Fluorescence images were obtained using a confocal microscope (LSM800; Carl Zeiss, Oberkochen, Germany) and fluorescence intensity were automatically analyzed by Zen software (Carl Zeiss, Oberkochen, Germany).
Microglia morphological analysis
Morphological analysis of microglia was performed using a method of MATLAB (version R2021a, The MathWorks Inc., Natick, MA, USA) as described previously33. Briefly, by tracking microglial soma and processes, intensity quantiles across the image were identified, and we then quantified the resultant image. To normalize fluorescence intensities between tissue samples, the quantile level of the background and the soma intensity were automatically modified. Minimum object recognition (i.e., soma) was set to 200–300 pixels. At 20× magnification, the total area of view (320 µm2) of the region of interest (hippocampal CA1 and DG) was evaluated.
Primary cell culture
E17-18 embryos and one-day-old C57BL/6 pups were used to culture primary glia and hippocampal neurons using a procedure described previously34, 35. In primary mixed glia culture, after removing the meninges from the cerebral hemisphere, tissue was dissociated into a single-cell suspension through gentle repetitive pipetting. Cells were then cultured in Dulbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum, l-glutamine, non-essential amino acids, and antibiotic/antimycotic in 75-cm2 flasks at 37°C in a 5% CO2 incubator, and the medium was changed every five days. For primary neuron culture, mice hippocampi were dissected and incubated in 2.5% trypsin buffer for 15 min. After being transferred to fresh culture media, hippocampi tissue was dissociated into a single-cell suspension through gentle repetitive pipetting. After counting, cells were cultured in neurobasal medium supplemented with l-glutamine, penicillin streptomycin, and B-27 supplements for 10 days before use for experiments.
3,3′-Diaminobenzidine (DAB) staining
DAB staining was conducted using published protocols with minor modification36. Briefly, hippocampal neurons were fixed with 4% paraformaldehyde and then incubated with antibodies for neuronal cell markers (NeuN, 1:1000, and MAP2, 1:1000, Millipore) after blocking. After incubation with biotinylated anti-rabbit-IgG antibody (Vector Labs, Burlingame, CA, USA) and streptavidin-horseradish peroxidase (HRP) conjugates (ThermoFisher Scientific, Waltham, MA, USA), the cells were treated with DAB (Sigma-Aldrich) for 30 min of reaction time.
Real-time
RT-PCR Total RNA was isolated from hippocampal tissue and cells using TRIzol reagent (Life Technologies, Grand Island, NY, USA), and 1-2 µg of total RNA were used for cDNA synthesis. Amplification of each gene was performed using an ABI Prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA) and SYBR Green PCR Master Mix (Applied Biosystems) as previously described37. Following the 2−ΔΔCt method38, relative mRNA levels were calculated, and delta-Ct values of each gene mRNA level were normalized to that of GAPDH. All real-time RT-PCR experiments were performed at least three times, and the mean ± s.e.m. values are presented unless otherwise noted. The following PCR primer sequences were used: GAPDH forward, 5′-AGG TCA TCC CAG AGC TGA ACG-3′; GAPDH reverse, 5′-CAC CCT GTT GCT GTA GCC GTA-3′; IL-1β forward, 5′-GTG CTG TCG GAC CCA TAT GA-3′; IL-1β reverse, 5′-TTG TCG TTG CTT GGT TCT CC-3′; IL-6 forward, 5′-CCA CGA TTT CCC AGA GAA CAT-3′; IL-6 reverse, 5′-TCC ATC CAG TTG CCT TCT TGG-3′; TNF-α forward, 5′-AGC AAA CCA CCA AGT GGA GGA-3′; and TNF-α reverse, 5′-GCT GGC ACC ACT AGT TGG TTG T-3′.
Enzyme-linked immunosorbent assay (ELISA)
The expression levels of IL-1β protein from mixed glia stimulated with S1 protein (5 µg/ml) for 6, 12, and 24 h were examined with an ELISA kit (R&D Systems Inc., Minneapolis, MN, USA) following the manufacturer’s protocols. The conditioned media from each time point was assessed in duplicate. Optical density was measured at 450 nm by a SpectraMax ABS Plus microplate reader (Molecular Devices, San Jose, CA, USA).
Statistical analysis
Statistical analyses were performed and graphs were made using GraphPad Prism 7.0 for Windows software (GraphPad Software Inc., La Jolla, CA, USA). The data were analyzed using an unpaired t-test as appropriate to assess significant differences. All data are expressed and plotted as the mean ± s.e.m. A p-value less than 0.05 was considered statistically significant. No animals or data point were excluded from the analysis. No statistical methods were used to pre-determine sample sizes, but our sample size is similar to those generally employed in the field.