Bioinformatic analysis

The gene list in the article for the three processes of efferocytosis, "find me", "eat me", and "engulf me", was created by combining references to gene lists associated with efferocytosis [21,22,23]. The data for the bioinformatic analyses were obtained from the public repository NCBI GEO (http://www.ncbi.nlm.nih.gov/geo), and datasets GSE30655 and GSE174574 were used in the study. The protein–protein interaction network (PPI) of differentially expressed genes (DEGs) associated with efferocytosis was generated using the STRING database and visualized using Cytoscape. The single-cell RNA-sequencing (scRNA-seq) dataset was visualized using UMAP plots.

Animals and groups

C57BL/6J mice were used in this study (male, aged 6–8 weeks, weighing 20–25 g), and were supplied by GemPharmatech Co., Ltd., Chengdu, China (license No. SCXK (Chuan) 2020-034). Mice were housed in well-ventilated cages under 12 h light/dark cycle at a temperature of 25 ± 1 °C and humidity of 50 ± 5% with adequate food and water. The mice were randomly and equally assigned to the sham, I/R, and EA groups, with the experimenters blinded to the groups. During the experiment, any mice that perished or did not meet the inclusion criteria were removed, and the number of mice in each group was replenished using the same modeling procedure. All animal experimentation procedures were approved by the Animal Ethics Committee of the Hospital of Chengdu University of Traditional Chinese Medicine (No.2023DL-022).

Mouse model of I/R

The I/R model was established using a modified Zea-Longa method [24]. We anesthetized the mice in an anesthesia induction box containing 5% isoflurane in oxygen/air mixture, then transferred them to an anesthesia mask to maintain anesthesia with 2% isoflurane (RWD Life Science, Shenzhen, China) from the animal anesthesia machine (R500, RWD Life Science, Shenzhen, China). The median neck muscle was divided, exposing the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). Litigation of the CCA proximal end and the ECA was performed permanently and ICA distal ends clamped with arterial clips to temporarily block blood flow to the brain. Then a monofilament (MSMC21B120PK50, RWD Life Science, Shenzhen, China) was placed inside the internal carotid artery by making an incision in the left common carotid artery to occlude the origin of the middle cerebral artery. MCAO for 60 min was followed by suturing of the wound to restore blood flow. During surgery, a heating pad was adopted to maintain the mice's body temperature at 37 °C. The sham group exposed only the left side of the vessel without monofilament insertion.

EA treatment

EA was given to the mice in the EA group 24 h after I/R. The mice were anesthetized using a mask with 2% isoflurane and then inserted with stainless acupuncture needles (0.18 mm × 13 mm, Huatuo, Suzhou, China) into the right ST36 (Zusanli, located longitudinally at 3 cun below the knee joint and intersecting the middle of the tibialis anterior muscle) and GV20 (Baihui, located at the intersection of the sagittal midline with the line between the ears). The parameters of the EA consisted of dispersive waves with a frequency of 2/15 Hz, an intensity of 1 mA, and a duration of five consecutive days with 20 min per day. The sham group and the I/R group received anesthesia only, without electroacupuncture.

Neurological deficit assessment

After 24 h of I/R in mice, neurological deficits were measured by an investigator who was blinded to the experimental groups. The specific scoring criteria are based on the Zea Longa five-point scale, as illustrated in Table 1 [24]. Mice that scored 1 to 3 points were considered successful in modeling and each mouse was scored daily for five consecutive days.

Infarct area measurement

At the end of the fifth day of neurological scoring, brain tissues were collected for 2,3,5-triphenyl tetrazolium chloride (TTC, Sangon Biotech, Shanghai, China) staining. The brain tissues were frozen at − 20 °C for 20 min. Each brain was cut into seven coronal sections at 1 mm intervals, then immersed in 2% TTC solution and incubated at 37 °C for 15 min protected from light, followed by 4% paraformaldehyde solution (Servicebio, Wuhan, China) immersion. The non-ischemic tissue showed a red color, while the infarcted areas were white. The infarct area and total area of the six sections were determined using ImageJ software, and the following formula was used to calculate the percentage of infarct area:( total infarct area/total area of slice) × 100%.

Histopathological observation

After 4% paraformaldehyde fixation, the brain containing hippocampal tissue was dehydrated, embedded in paraffin, and coronally sectioned. Sections were stained with Nissl staining solution (Servicebio, Wuhan, China) and Hematoxylin–Eosin staining kit (Servicebio, Wuhan, China), respectively. A light microscope (Nikon Eclipse E100, Nikon, Tokyo, Japan) was used to examine the pathological changes of cortex and hippocampus tissues.

Multiplex immunofluorescence staining

Paraffin-embedded sections were washed in distilled water after they had been deparaffinized. The antigen was repaired using a microwave with ethylene diamine tetraacetic acid (EDTA, PH8.0, C1033, Solarbio, Beijing, China) antigen repair buffer. Sections were pretreated with 3% H2O2 for 10 min to block endogenous peroxidase activity and incubated in 3% (w/v) bovine serum albumin-V (BSA-V, A8020, Solarbio, Beijing, China) in Phosphate Buffered Saline (PBS, Servicebio, Wuhan, China) for 30 min at room temperature (RT). The following antibodies were used as primary antibodies at 1:200 dilution in PBS overnight at 4 °C: anti-Iba1 (ab178846, Abcam, Cambridge, UK), anti-CD206 (GB113497-100, Servicebio, Wuhan, China), anti-NeuN (A19086, ABclonal, Wuhan, China), and anti-Abca1 (A21976, ABclonal, Wuhan, China). Wash with PBS and incubate with anti-rabbit IgG antibody 1: 400 (FCMCS, FMS-Rb01) for 50 min at RT in the dark. The following fluorescent dyes were applied to the sections: IF488-Tyramide (G1236-1, Servicebio, Wuhan, China), IF555-Tyramide (G1236-2, Servicebio, Wuhan, China), and IF647-Tyramide (G1236-3, Servicebio, Wuhan, China). Incubations were shielded from light for 10 min. Multiplex staining was repeated in series for each staining step, and antigen repair was performed between each staining step. The sections were subsequently incubated with 4′,6-diamidino-2-phenylindole (DAPI, G1236-5, Servicebio, Wuhan, China) for 10 min at RT in the dark, washed, and sealed with an anti-fluorescence quenching sealer (G1401-5, Servicebio, Wuhan, China) finally. These brain sections were observed with Nikon Eclipse E100. ImageJ software was used to count the number of positive cells.

Western blot

The hippocampus and parietal cortex were extracted from the mouse brain and weighed, and a BCA protein assay kit (BL521A, Biosharp, Beijing, China) was used to determine protein concentration. Each sample contained 15–50 μg of protein, which was electrophoresed on a sodium dodecyl sulfate–polyacrylamide gel and then transferred to PVDF membranes (IPVH00010, 0.45 µm, Millipore, Immobilon, Ireland). The membranes were blocked with 5% (w/v) skimmed milk in tris buffered saline with Tween-20 (TBST) buffer for 2 h. After washing with TBST, the membranes were incubated at 1:1000 dilution in TBST overnight at 4 °C with the following antibodies: anti-Iba1 (ab178846, Abcam, Cambridge, UK), anti-CD206 (A8301, ABclonal, Wuhan, China), anti-NeuN (A19086, ABclonal, Wuhan, China), anti-Abca1 (A21976, ABclonal, Wuhan, China), anti-GAPDH(GB12002, Servicebio, Wuhan, China). After washing with TBST, the secondary antibody diluent of the corresponding species of the primary antibody, goat anti-rabbit IgG antibody (FMS-Rb01, FCMCS, Nanjing, China) or mouse (FMS-MS01, FCMCS, Nanjing, China), was added and incubated for 1.5 h at RT. An enhanced chemistry luminescence (ECL, BL520A, Biosharp, Beijing, China) method was used to detect protein bands after the membranes were again washed. The intensity of the bands was analyzed using ImageJ software.

RNA extraction and RT-qPCR analysis

The cortex and hippocampus of the mice were promptly flash-frozen in liquid nitrogen and stored at − 80 °C after extraction. Total RNA was extracted using the MolPure Cell/Tissue Total RNA Kit (Yeasen Biotechnology, Shanghai, China), and purity and concentration were determined using the Nanodrop 2000 UV–visible spectrophotometer. RNA is reverse transcribed to cDNA using Hifair III 1st Strand cDNA Synthesis SuperMix for qPCR (Yeasen Biotechnology, Shanghai, China). Subsequently, RT-qPCR analysis was conducted utilizing the primers listed in Table 2, based on the Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (Yeasen Biotechnology, Shanghai, China). For detailed procedures, please refer to the corresponding kit instructions. The primer sequences utilized for amplification are displayed in Table 2, provided by Sangong Biotech (Sangong Biotech, Shanghai, China).

Table 2 RT-qPCR primer sequencesStatistical analyses

All data were analyzed using either GraphPad Prism v.9.5.0 or R software and expressed as mean ± standard error of the mean. Survival curves were compared using the Log-rank test. Tukey's multiple comparisons test was used to test for differences in percentage weight change and Neurological Deficit. Other data that followed a normal distribution with homogenous variance were analyzed using One-way analysis of variance for multiple comparisons. P-values less than 0.05 were deemed statistically significant.