Hanks balanced salt solution (hbss)

Six 120-days-old female C57BL/6 and C57BL/6 (AD model) specific pathogen-free mice were obtained from the Guangdong Medical Experimental Animal Center (License No. SCXK 2018-0002; Guangzhou, China) and placed in a polystyrene cage in a room with constant temperature (23±2 ℃) and humidity (45%±15%) conditions, a 12-hour light/dark cycle, and ad libitum access to standard food and water. After a week of adaptive feeding, the mice were euthanized by intraperitoneal injection of sodium pentobarbital (150 mg/kg body weight). Thereafter, the brain tissue was aseptically removed, the cerebral cortex was carefully opened, the hippocampus was exposed, and the surrounding tissues of the hippocampus were separated using ophthalmic scissors and placed into Hank’s balanced salt solution (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). A protocol was prepared before the study without registration. Animal experiments were approved by the Animal Care and Use Committee of Guangxi Medical University (approval No. 2017JJB10090) and were conducted in accordance with the National Institutes of Health guidelines for the care and use of animals.

At 24 h following transfection, THP-1 cells were washed with Hank's balanced salt solution (Beijing Solarbio Science & Technology Co., Ltd.) before the culture medium was replaced with fresh RPMI-1640 medium. A final concentration of 1 µg/ml LPS (Sigma-Aldrich; Merck KGaA) was subsequently added into the culture medium, followed by incubation for a further 3 h. The cells were then collected for the measurement of IRAK1 and p68 expression, whilst the supernatant of the culture medium was collected for the measurement of cytokine levels, as described previously (15 (link)). The cells were divided into the following experimental groups: Blank control, no RNA transfection or LPS treatment; LPS group, treated with LPS only; mimic group, miR-146 amimic+LPS; NC mimic group, NC mimic + LPS; inhibitor group, miR-146a inhibitor + LPS; and inhibitor NC group, inhibitor NC + LPS.

The Sox10-creER mice were crossed with Rosa26 tdTomato mice to obtain Sox10/tdTomato double-positive (Sox10/tdTomato) littermates. The Sox10/tdTomato mice were treated with a single injection of 75 mg/kg tamoxifen (Sigma, USA) intraperitoneally at postnatal day (P) 0 to induce the Cre recombinase system and activate tdTomato fluorescence, and were sacrificed at P3. Briefly, after decapitation, the skulls of mice were cut along the midsagittal plane from the foramen magnum, and the temporal bones were placed in Hank's Balanced Salt Solution (Solarbio, China). The cochlear capsule was removed by fine forceps to expose the membranous labyrinth under a dissecting microscope, and the stria vascularis and the organ of Corti were removed the modiolus with the spiral ganglion was taken out. Collagenase type Ⅳ (1.5 mg/mL, Sigma) was used to digest the modiolus at 37 °C for 5 min, and fetal bovine serum (Gibco, USA) was added to terminate digestion. Culture medium was added to suspend the cells after centrifugation and pipetted up and down approximately 80–100 times to disrupt the tissue into single cells, and a 40-μm cell strainer (Corning, USA) was used to filter the single-cell suspension. Finally, dissociated cells from the ganglia were sorted on a BD FACS Aria III (BD biosciences, USA), and tdTomato+ and tdTomato− cells were collected separately.