Animal experimentation
Male C57BL/6 mice (6–8 weeks old, 18–20 g) were purchased from the Experimental Animal Center, Zhejiang Academy of Medicine Sciences, Hangzhou, China (Certificate No. SYXK Zhe 2019-0011). Mice were housed at 22 ± 2℃, relative humidity 50 ± 10%, and a 12-h light/dark cycle. They were allowed to have free access to water and food. All experiments were carried out following the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The number of animals used in the study and their suffering were minimized. The experimental protocols were approved by the Ethics Committee of Laboratory Animal Care and Welfare, School of Medicine, Zhejiang University, Hangzhou, China.
PLX 3397 treatment
For pharmacological ablation of microglia in mouse gavage experiments, a working solution of pexidartinib (PLX3397, GLPBIO, CA, USA) was made in 10% DMSO (Solarbio.BeiJing, China) and 40% PEG300 (Solarbio.BeiJing, China) in water to obtain a final concentration of 10 mg/ml. Mice were administered with PLX3397 via oral gavage at a dosage of 80 mg/kg/day every day for 3 consecutive weeks, as described previously (formulated in AIN-76A standard irradiated chow by Research Diets at a dose of 290 mg/kg for PLX3397)[13]. Mice in the control group were treated with the same volume of vehicle (normal saline).
Sepsis-associated encephalopathy (SAE) model
The SAE mouse model was established as described previously, i.e., via the cecal ligation and puncture (CLP)[14]. Briefly, mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg/kg). After disinfection, an incision of 2–3 cm was made at 1.5 cm below the xiphoid to expose the abdominal cavity. The cecum was isolated and ligated at half the distance between the distal pole and the base of the cecum and punctured with a 22-gauge needle. Fecal contents were gently extruded into the peritoneal cavity. Then, the cecum was carefully placed back into the peritoneal cavity, and the abdomen was sutured. After surgery, mice were returned to their cages with a warm cotton pad and free access to food and water. For sham mice, the abdominal cavity was opened to expose the cecum without ligation or puncture. The neurobehavioral assessment was determined by a modified neurologic severity score (mNSS)[15]. SAE was defined as having a score ≥ 7.
Experimental groups
Mice were randomly divided into four groups: sham (vehicle), SAE, vehicle + PLX3397, and SAE + PLX3397. Mice in the four groups were treated with either vehicle or PLX3397 by oral gavage. On the 21st day after the PLX3397 administration, mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg/kg) for the SAE procedure. At the end of the experiment (day 23), mice were re-anesthetized and perfused with saline followed by 4% paraformaldehyde (PFA). Brains were removed, fixed in 4% PFA for 24 hours, and transferred to 30% sucrose solution for 3 days. Next, 20-µm sections from the frontal to the occipital poles were cut by cryomicrotome (Leica CM1950, Wetzlar, Germany) and were further used for immunostaining. For biochemical analysis and immunoblotting, the brains were collected and stored at -80℃.
Primary cortical neuron culture
Primary hippocampal neurons were obtained from the cortex of neonatal Sprague Dawley (SD) rats (Experimental Animal Center, Zhejiang Academy of Medicine Sciences, Hangzhou, China). Briefly, the cortex of neonatal SD rats was dissected and digested with 0.25% EDTA-free trypsin at 37℃ for 10 min. The dissociated cells were immediately seeded onto poly-l-lysine pre-coated flasks and incubated in high glucose DMEM containing 10% fetal bovine serum (FBS), 10% horse serum, 2 mM glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin for 24 h in a humidified atmosphere of 5% CO2/ 95% air at 37℃ (standard cell culture conditions). After 24 h, the medium was changed to high glucose DMEM containing 5% horse serum, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.04% B27, and 0.01% N2. On day 3, 10 µM cytosine arabinoside was added for 24 h to inhibit the growth of glial cells in cultured neurons; the medium was refreshed every 3 days. On day 10, > 95% of the cultured cells were neurons, as determined by immunofluorescent staining for microtubule-associated protein-2 (MAP2).
Cell culture
Mouse BV2 microglial cells, mouse HT-22 hippocampal neurons cells, rat PC12 adrenal medulla pheochromocytoma cells, and human SH-SY5Y neuroblastoma cells were obtained from the Chinese Academy of Medical Sciences (Beijing, China). BV2, HT-22, and PC12 lines were cultured in RPMI-1640 medium supplemented with 10% FBS, 5 U/ml penicillin, and 5 µg/ml streptomycin under standard cell culture conditions. SH-SY5Y line was cultured in 43% Ham’s F-12 medium and 43% MEM basal medium supplemented with 10% FBS, 1% MEM NEAA, 1% sodium pyruvate, 1% GlutaMAX-1, 5 U/ml penicillin, and 5 µg/ml streptomycin under standard cell culture conditions. To avoid the activation of BV2 cells and microglia, FBS was heat inactivated for 30 min at 56℃ before use. Before stimulation, cells were seeded in 5x10^5 cells per well in a tissue culture-treated 6-well plate (Corning, NYC, US) and grown to 80% confluency. For the experiments, BV2 cells were pretreated with 1 µg/ml LPS (Sigma-Aldrich, MO, USA) for 18 hours to stimulate LD formation. The cells were treated with TLR4 inhibitor 1uM/L TAK-242 (GLPBIO, MFEE, USA).
Conditioned medium (CM) treatment
To evaluate the effects of the microglial CM on primary neurons, HT-22, PC12, or SH-SY5Y cells, the supernatant was collected from the BV2 line pretreated with 1 µg/ml LPS. The CM was collected and centrifuged at 6,000 rpm at 4°C for 10 min to remove cell debris. Primary hippocampal neurons, HT-22, PC12, or SH-SY5Y cells were incubated with the CM from the microglia for 18 hours. Subsequently, cell viability and pyroptosis were assessed.
Transwell co-culture system
RPMI-1640 supplemented with 10% FBS, 5 U/ml penicillin, and 5 ug/ml streptomycin was used as a medium. To avoid the activation of BV2 cells and microglia, FBS was heat inactivated for 30 min at 56℃ before use. HT-22 cells plated on a coverslip were transferred into the lower compartment of a 6-well Transwell system and BV2 microglia were plated (10^6 cells per well) onto the insert of the Transwell (0.4 mm pore-size polycarbonate membrane precoated with poly-l-lysine).
Cell viability determination
Cell viability was determined using Cell Counting Kit-8 (CCK-8; Beyotime Technology Inc., Shanghai, China) according to the manufacturer's instructions. Cells were seeded into a 96-well plate and treated with CM in the presence or absence of LPS. After treatment, the cells were rinsed with phosphate buffer saline (PBS) and incubated with 10 µl CCK-8 solution for 1.5 h at 37℃. Optical density was measured at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). Data were expressed as % of controls.
Immunostaining
To determine the localization of LDs in different cell types, immunostaining was performed. The 20-µm brain sections were rinsed with 0.01 M PBS, incubated for 1 h with 5% goat serum (Zhongshan Biotechnology Co., Beijing, China) supplemented with 0.3% Triton X-100 to block nonspecific IgG binding, and then incubated overnight at 4℃with the following primary antibodies: rabbit anti-MAP2 (1:200; Affinity Biosciences), mouse anti-neuronal nuclear antigen (NeuN, 1:500, Millipore, Billerica, MA, USA),rabbit anti-ionized calcium-binding adapter molecule 1 (IBA1, 1:500, Wako Pure Chemical Industries, Ltd.), mouse anti-lysosome-associated membrane protein 2 (LAMP2, 1:200, Proteintech), mouse anti-cleaved caspase 1 (1:200, Affinity Biosciences), mouse anti-caspase 1 (1:200, Affinity Biosciences), and anti-LC3A/B (1:200, Affinity Biosciences). Sections were then incubated with DyLight 549- or 488-conjugated secondary antibody (1:200, EarthOx SF, USA) for 2 h at room temperature. Nuclei were stained with DAPI.
Cells seeded on coverslips in 24-well plates were fixed in 4% PFA for 20 min, permeabilized with 0.5% Triton X-100 for 10 min, and blocked with 5% goat serum for 30 min at 37℃. Cells were treated overnight at 4℃ with the following primary antibodies: anti-MAP2 (1:200), anti-LAMP2 (1:200), anti-cleaved caspase 1 (1:200), anti-caspase 1 (1:200), and anti-LC3A/B (1:200) followed by DyLight 549- or 488-conjugated secondary antibody (1:200) for 2 h at room temperature. For negative control, PBS was used instead of the primary antibody. Nuclei were stained with DAPI. After mounting with an Anti-Fade Mounting Medium (Beyotime Technology Inc., Shanghai, China), images were taken with the Olympus IX71 fluorescence microscope equipped with a DP71 digital camera using Ocular software. Eight images of nonoverlapping fields for each site in the same sections (40x magnification) were randomly taken, and the average area of LDs (µm2) per view field and the average number of LDs per cell was calculated using ImageJ software.
BODIPY staining
4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503, Invitrogen; Thermo Fisher Scientific, Inc.) fluorescent neutral lipid dye was used to visualize LDs in cells or brain sections by fluorescent microscopy. The 20-µm brain sections or fixed cells on coverslips in 6- or 24-well plates were rinsed with 0.01 M PBS and then incubated for 30 minutes with BODIPY 493/503 at 37℃. After mounting with an anti-fade medium, the stained samples were examined under the Olympus IX71 fluorescence microscope. Eight images of nonoverlapping fields for each site in the same sample (40x magnification) were randomly taken, and Image J software was used to determine the per-cell number of lipid vesicles for each view field.
Immunoblotting
The brain tissue or total cell protein extraction was performed using ice-cold RIPA buffer (Beyotime Technology Inc., Shanghai, China) according to the manufacturer's instructions. The homogenates were centrifuged at 12,000 x g at 4℃ for 30 min, and the supernatant was used for subsequent analyses. Equal amounts of protein (60 µg) for each sample were separated by 15% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked in 5% bovine serum albumin (BSA), and sequentially incubated with primary antibodies: anti-IBA1 (1:500), anti-LAMP2 (1:200), anti-cleaved caspase 1 (1:200), anti-caspase 1 (1:200), anti-LC3A/B (1:200), and mouse anti-glyceraldehyde-3- phosphate dehydrogenase (GAPDH, 1:5000, Kangchen Technology Inc., Shanghai, China). Next, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (1:200, Thermo Fisher Scientific) for 2 h at room temperature. Proteins were visualized using chemiluminescent reagents according to the manufacturer’s instructions (Merck, Millipore) with a Bio-Rad ChemiDoc Touch Imaging System and quantified with Quantity One software (Bio-Rad, Hercules, CA, USA).
Assessment of cell pyroptosis
Cell pyroptosis was measured using FLICA 660 Caspase-1 Kit (MyBioSource, VAN, CAN) according to the manufacturer’s instructions[16]. Briefly, after 18 h treatment with LPS, cells were collected, washed once with cold PBS, and centrifuged at 2,000 ×g for 5 min. The pellet was resuspended in 300 µl of binding buffer containing active caspase 1-FITC and PI solution and then stained in the dark for 15 min at room temperature. The percentage of cell pyroptosis was determined using a FACSCalibur cytometer (Beckman Coulter CytoFLEX S, CA, USA). The fold increase in cell death rate was normalized to that of the control group.
Measurement of lactic acid, triglyceride, and cholesterol levels
Lactic acid, triglyceride, and cholesterol levels were measured according to the manufacturer’s instructions (Triglyceride assay kit, Total cholesterol assay kit, Lactic acid assay kit; Jiancheng Technology Inc., Nanjing, China). After 18 h stimulation with CM, the brain tissue homogenates, cultured cell supernatants, or cell lysates were collected and used for the subsequent analysis of lactic acid, triglyceride, or cholesterol concentrations. The values were calculated by measuring the absorbance at 500 nm (CE), 510 nm (TAG), and 530 nm (lactic acid) using a multifunctional enzyme standard in a multimode plate reader (Tecan Spark, Tecan, Switzerland).
Statistical analysis
All data were expressed as means ± standard error of the mean (SEM). Student's t-test was used to compare statistical differences between two groups and one-way ANOVA followed by Tukey's test was applied for multiple comparisons. p < 0.05 was considered a statistically significant difference.