2.1 Animals and tMCAO models
Male C57BL/6 mice (6–8 weeks old, weight 20 ± 3 g) were procured from Liaoning Changsheng Biotechnology Co., Ltd and housed in a hygienic and comfortable habitat under a 12-h natural light-dark cycle to simulate their circadian rhythm. The Animal Experiment Center of Harbin Medical University provided adequate food and water. The animals were randomly divided into four groups (n = 4/group): sham group, 24h-tMCAO group, DSF + 24h-tMCAO group, and vehicle + 24h-tMCAO (dimethyl sulfoxide < 2%). The DSF + 24h-tMCAO group was intraperitoneally administered DSF (50 mg/kg, twice daily), starting from the day before the surgery and continuing postoperatively. The mice were anesthetized with 2% isoflurane, and body temperature was maintained at ~ 37°C throughout the surgical and postoperative periods using a servo-controlled heating blanket. Focal cerebral ischemia was achieved by occluding the middle cerebral artery (MCA). After performing a midline incision in the neck, a doccol suture (602256PK5Re; US) was inserted into the right external carotid artery and then reversed into the right internal carotid artery via the common carotid artery to occlude the blood supply to the MCA. The sutures were then removed after 1 h.Following suture removal, the mice exhibited recovery times of 6 h, 24 h, 3 d, 5 d and 7 d.Mice died of cervical dislocation after anesthesia at the above time points. This experiment was reviewed and approved by the ethical review committee of Harbin Medical University. (IACUU NO.2022133).The management and use of mice are consistent with the relevant guidelines of US National Institutes of Health.
2.2 2,3,5-Triphenyltetrazolium chloride (TTC) staining
Briefly, the harvest mouse brain was sliced into seven pieces (1 mm thick) from the rostral end of the frontal lobe. Cells were then incubated in TTC (Solarbio, Beijing, China), shielded from light for 30 min, and fixed in 4% paraformaldehyde. Planar infarct volume measurements were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
2.3 Hematoxylin-eosin and Nissl stainings
The specimens were fixed in a 4% paraformaldehyde solution and subsequently embedded in paraffin. Histological sections (4 µm thick) were prepared and stained with hematoxylin-eosin. For Nissl staining, a 1% toluidine blue solution was heated to 60°C, followed by the immersion of all sections for 40 min. Subsequently, sections were washed with 70% ethanol and rapidly differentiated using 95% ethanol. The tissue sections were examined and imaged under a light microscope (Nikon, Tokyo, Japan).
2.4 Immunofluorescent staining
Briefly, the prepared brain tissues were sequentially immersed in a 4% paraformaldehyde solution and 30% sucrose solution for 48 h. After embedding in optimal cutting temperature compound and freezing, frozen blocks were sectioned (7-µm thick slices) using a freezing microtome. Sections were treated with a blocking solution (Beyotime, China) containing Triton X-100 for 20 min and incubated overnight at 4°C with the following primary antibodies: ASC (sc-514414, 1:50 dilution, Santa Cruz, USA), Caspase-1 (sc-56036, 1:50 dilution, Santa Cruz, USA), GSDMD (sc-393581, 1:50 dilution, Santa Cruz, USA), HSP70 (T55496, 1:50 dilution, Abmart, China). Following hydrations with PBST (3×), the samples were incubated with the appropriate fluorophore-conjugated secondary antibody (Boster, China) for 1 h. Nuclei were stained with 30µL DAPI (Abcam, UK) to ensure no bubbles. After 10 min, the slides were examined using light microscopy.
2.5 Immunohistochemical staining
The fixed brain tissues were embedded in paraffin, sectioned, deparaffinized, and subjected to antigen retrieval using a citrate solution after blocking with 3% hydrogen peroxide. The sections were then blocked with fetal bovine serum for 1 h. Subsequently, the sections were incubated overnight at 4°C with primary antibodies against FDX1 (T510671,1:200 dilution, Abmart, China), DLST (TD13671,1:200 dilution, Abmart, China), ATP7A (PA7106, 1:200 dilution, Abmart, China), ATP7B (TA0410, 1:200 dilution, Abmart, China), Caspase-1 ( 22915-1-AP, 1:200 dilution, Proteintech, USA), and IL-18 (10663-1-AP, 1:200 dilution, Proteintech, USA), followed by subsequent incubation with secondary antibodies (Boster, China) for 1 h at room temperature. Color development was achieved using diaminobenzidine. Then, sections were stained with hematoxylin. Subsequently, the sections were dehydrated and sealed before imaging under a light microscope at 200× magnification. The acquired images were analyzed using ImageJ software to determine the average density within the observation area.
2.6 Cu level detection
Cu levels were detected using colorimetric quantitative kits provided by Nanjing Jiancheng BioEngineering Institute (Jiangsu, China). The right brain tissue was measured, followed by the addition of double-distilled water at a ratio of 1:9 and supernatant collection by ultrasonic centrifugation. For protein quantification, the absorbance (580 nm) was measured using a microplate reader (MD, Shanghai, China) after the sequential addition of reagents.
2.7 Western blot analysis
Brain tissue from the right hemisphere was lysed (RIPA: protease inhibitor: phosphatase inhibitor, 100:1:1; lipoacylated protein was added in an equal proportion of TCEP). To determine the sample protein concentration, the supernatant was denatured by boiling, using the BCA protein assay kit (Beyotime, China). Proteins were subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis and subsequently transferred onto polyvinylidene fluoride membranes using the sandwich method. Then, the membranes were blocked with 5% skim milk for 1 h at room temperature, followed by overnight incubation with primary antibodies at 4°C in a refrigerator. The membranes were incubated with goat anti-mouse and anti-rabbit secondary antibodies (Abmart, China) at room temperature for 60 min. Next, the membranes were washed with Tris-buffered saline with 0.1% Tween® 20 detergent and incubated with an enhanced chemiluminescence reagent (Biosharp, China) for detection. The primary antibodies employed were as follows: GSDMD (EPR20859, 1:1000, Abcam, UK); Caspase-1(22915-1-AP, 1:1000 dilution, Proteintech, USA), ASC (sc-514414, 1:500, Santa Cruz, USA),IL-1β(RM1009, 1:1000, Abcam, UK), IL-18 (10663-1-AP, 1:1000 dilution, Proteintech, USA), IL-17(26163-1-AP, 1:1000, Proteintech,USA),TLR4(66350-1-Ig, 1:1000dilution, Proteintech, USA),NLRP3(EPR23073-96, 1:1000, Abcam, UK),TNFα(60291-1-Ig, 1:1000 dilution, Proteintech, USA), LIAS(67298-1-Ig, 1:1000 dilution, Proteintech, USA), NF-κB(66535-1-Ig, 1:1000 dilution, Proteintech, USA) and SLC31A1 (CTR1), ATP7B (TA0410, 1:1000 dilution, Abmart, China), HSP70(T55496, 1:1000 dilution, Abmart, China), FDX1(T510671, 1:1000 dilution, Abmart, China), SDHB(EPR10880, 1:50000, Abcam, UK), DLAT(T58125, 1:1000 dilution, Abmart, China), and DLST (TD13671, 1:1000 dilution, Abmart, China). For protein bands, gray values were quantified using ImageJ software.
2.8 Transmission electron microscopy
Brain tissues were cut into cubes (≤ 1 mm3) in 2.5% glutaraldehyde solution. After fixation with osmium tetroxide, tissue samples were subjected to repeated dehydration using acetone, followed by the addition of resin overnight at room temperature. The resulting tissue sections (70 nm) were stained with uranyl acetate and lead citrate and dried for transmission electron microscopy.
2.9 Statistical analyses
All results are presented as the mean ± standard error of the mean. Data were analyzed using analysis of variance, followed by Tukey's post-hoc test. Statistical analyses were performed using GraphPad Prism version 9.5 (GraphPad Software, Inc., Boston, MA, USA). Statistical significance was set at P < 0.05.