Animal Treatment and Surgery

Male C57BL/6 mice, aged 18 months and weighing 35–45 g, were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals were housed in a specific-pathogen-free (SPF) barrier facility and maintained on a 12-h light/dark cycle, with an ambient temperature of 22 ± 2 °C and a relative humidity of 60 ± 5%. All the experimental procedures adhered to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and received approval from the Institutional Animal Care and Use Committee of Shandong Second Medical University (approval no. 2023SDL376). Every effort was made to minimize animal suffering and reduce the number of animals utilized.

A rodent model of PND was established through exposure of the right common carotid artery under sevoflurane anesthesia, as previously described [16]. Induction occurred in an anesthesia chamber filled with 7–8% sevoflurane, with maintenance at 3% sevoflurane throughout the surgical procedure. Following confirmation of the absence of a response to painful stimuli, the cervical region was shaved and disinfected. A 1.5 cm longitudinal midline incision was made in the neck, soft tissues over the trachea were gently retracted, and the right common carotid artery was carefully isolated from adjacent structures over a length of 1 cm, ensuring that there was no damage to the vagus nerve. The wound was irrigated and closed with surgical sutures, and postoperatively, the incision site was infiltrated with 0.5% lidocaine to alleviate pain. Throughout anesthesia, body temperature was maintained at 37 °C using a heating pad; the surgical procedure lasted approximately 15 min, with the total duration of anesthesia being 2 h. After surgery, the animals were placed in a 37 °C incubator until full spontaneous recovery was achieved.

Cell Culture and Treatment

The BV-2 mouse microglial cell line was purchased from Cobioer Biological Technology Company (RRID:CVCL_0182, Nanjing, China). BV-2 cells were cultured in RPMI-1640 medium (11875085, Gibco, Shanghai, China) supplemented with 10% fetal bovine serum (10099141C, Gibco, Shanghai, China) and 1% penicillin–streptomycin (SL6040, Coolaber, Beijing, China) at 37 °C under a humidified atmosphere of 5% CO2. BV-2 microglia were randomly divided into three groups: a control group, a lipopolysaccharide (LPS) (1 µg/mL, L8880, Solarbio, Beijing, China) group, and a PK11195 (0.5 µM, CO424, Sigma‒Aldrich, USA) + LPS group. In the PK11195 + LPS group, the cells were pretreated with PK11195 for 1 h prior to LPS exposure, which was maintained for 24 h. After the treatment period, the cells were harvested for further analysis.

Open Field Test (OFT)

On perioperative Day 4, the spontaneous activity of the mice was evaluated in an open field (40 × 40 × 40 cm) to assess their exploratory behavior. Each mouse was placed in the center of the arena and allowed to roam freely for 5 min. Their activities were automatically recorded using a video tracking system. The total distance traveled by the mice, as well as the time spent exploring the center versus the corners of the arena, was recorded.

Novel Object Recognition (NOR) Test

The object recognition test was conducted in a square chamber measuring 40 × 40 × 40 cm, which was constructed from black, perforation-free plastic. The procedure included a training phase and a testing phase. On perioperative Day 5, mice were permitted to explore two identical objects for a duration of 5 min. On perioperative Day 6, one of the objects was replaced with a new object of a different shape, and the mice were given an additional 5 min to explore both objects. The exploration time was recorded as the duration the mice spent sniffing or touching the objects with their noses and/or forepaws at a distance of less than 2 cm. The entire behavioral process was captured using a video tracking system (VisiTrack). Cognitive function was assessed through the novel object recognition rate, also referred to as the recognition index, which was calculated as the ratio of exploration time spent on the new object to the total exploration time. To eliminate olfactory cues, the area was cleaned with a 75% ethanol solution.

Y-maze Test (Y-maze)

The Y-maze consisted of three cubic arms, each measuring 30 cm × 6 cm × 15 cm (length × width × height), positioned at 120° angles to one another against a white background. The entire behavioral testing process was recorded using a video tracking system (VisiTrack). The spontaneous alternation test took place on perioperative Day 7, following the completion of the open field and novel object recognition tests by the mice. The basic procedure involved providing a single mouse with free access to the three arms of the maze for 8 min, during which the sequence of arm entries was documented. From these sequences, the proportion of entries into arms distinct from the previously visited one was calculated to derive the spontaneous alternation behavior (SAB) score. The formula is as follows: SAB = [(ABC + ACB + BAC + BCA + CAB + CBA)/(A + B + C−2)] × 100%.

Serum Copper Levels

Copper levels in the serum were measured using a colorimetric assay kit (E-BC-K300-M, Elabscience, Wuhan, China). A total of 20 µL of serum was added to the wells of a 96-well plate, followed by the addition of 300 µL of the detection reagent mixed with the samples. The plate was then incubated at 37 °C for 5 min, after which the optical density was assessed at 580 nm using a microplate reader.

Copper Levels in Hippocampal Tissue and Cells

Hippocampal tissue was accurately weighed and transferred into a microwave digestion vessel. Five milliliters of ultrapure concentrated nitric acid was added, and the mixture was vortexed for 1 min, and predigested at 120 °C for 20 min. After cooling to room temperature, an additional 5 mL of concentrated nitric acid was introduced, and the sample was subjected to a programmed microwave digestion protocol. Upon completion, the digest was evaporated at 180 °C until the volume was reduced to 1 mL and the solution appeared clear and colorless. After returning to ambient temperature, the residue was quantitatively transferred into a 50-mL polypropylene centrifuge tube and brought to the final volume with ultrapure water in three sequential rinses. The solution was vortexed for 1 min, passed through a 0.22 µm membrane filter, and analyzed for copper content using inductively coupled plasma–mass spectrometry (ICP-MS; PE NexION 300D, PerkinElmer, Waltham, MA, USA).

Western Blot Analysis

Cells that underwent various treatments were lysed in RIPA buffer (R0010, Solarbio, Beijing, China) supplemented with PMSF (P0100, Solarbio, Beijing, China). The protein concentration was quantified with a BCA protein assay kit (PC0020, CWBIO, Beijing, China), and the samples were heated at 100 °C for 5 min. Each group of 20 µg protein samples was resolved on 8%, 10%, or 12% SDS‒PAGE gels, as appropriate, followed by transfer onto PVDF membranes (IPVH00010, 0.45 µm, Millipore, Germany). The membranes were blocked with 5% skim milk in TBST buffer for 3 h and subsequently incubated with primary antibodies overnight at 4 °C. An overview of the primary antibodies used is provided in Table 1. Next, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (SA00001-1, goat anti-rabbit, 1:7000; Proteintech, Wuhan, China) at room temperature for 90 min. Protein expression levels were assessed using an enhanced chemiluminescence detection system (Tanon 4600, Shanghai, China).

Table 1 Primary antibodies used for western blottingQuantitative Real‑Time PCR (qRT‑PCR)

Primers were synthesized by BGI Tech Solutions (Beijing, China). Total RNA was extracted using TRIzol reagent (R401-01, Vazyme, Nanjing, China) following the manufacturer’s instructions and subsequently reverse-transcribed into cDNA using the HiScript® III RT SuperMix Kit (R323-01, Vazyme, Nanjing, China). Amplification reactions were conducted in triplicate with the 2 × ChamQ SYBR® qPCR Master Mix Kit (Q312-02, Vazyme, Nanjing, China). The mRNA expression levels were normalized to those of β-actin and assessed according to the fold change. Data analysis was performed using the 2−ΔΔCT method. The primer sequences can be found at Table 2.

Table 2 Primer sequences for qRT‒PCRImmunofluorescence (IF)

Following treatment, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min. The cells were then blocked with 5% goat serum in PBS (P1020, Solarbio, Beijing, China) for 1 h at room temperature and incubated overnight with primary antibodies at 4 °C. The next day, after the cells were washed three times with PBS, they were incubated in the dark at 37 °C for 2 h with goat anti-rabbit IgG (SA00013-2, 1:500; Multi Sciences, Hangzhou, China) or goat anti-mouse IgG (SA00013-3,1:500; Multi Sciences, Hangzhou, China) secondary antibodies. Finally, fluorescence images were captured using a fluorescence microscope (Olympus, Tokyo, Japan) and analyzed with ImageJ. The mean fluorescence intensity was background-corrected and normalized to the number of DAPI-positive nuclei per field.

Enzyme-Linked Immunosorbent Assay (ELISA)

Levels of tumor necrosis factor (TNF-α) in murine hippocampal tissue were quantified using enzyme-linked immunosorbent assay following the manufacturer’s specifications (SEKM-0034, Solarbio, Beijing, China).

Cell Viability Assay

Cell viability was measured by the Cell Counting Kit-8 (CCK-8) assay (CK001, LABLEAD, Beijing, China). Logarithmically growing BV-2 microglia were seeded into 96-well plates and incubated with 100 μL of complete medium per well. The cells were incubated for 24 h at 37 °C under 5% CO2 to facilitate adherence and recovery. Following this period, the medium was replaced with fresh medium containing 1 µg/mL LPS, and the incubation continued for an additional 24 h. Next, 10 μL of CCK-8 reagent was added to each well, and the plates were gently agitated before being incubated for 30 min at 37 °C in the dark. The absorbance at 450 nm (A450) was measured using a microplate reader, with blank medium serving as the zero reference. Cell viability (%) was calculated using the formula [(A450 treated − A450 blank)/(A450 control − A450 blank)] × 100%.

Reactive Oxygen Species Detection

Intracellular superoxide anion levels were detected using dihydroethidium (DHE). BV-2 cells were induced by LPS, with or without PK11195 pretreatment, followed by three washes with PBS. The cells were then incubated in the dark at 37 °C with 2.5 µM DHE (S0063; Beyotime, Shanghai, China) for 30 min. After the cells were washed three times, images were captured using a fluorescence microscope (Olympus, Tokyo, Japan) and analyzed with Image-Pro Plus.

Mitochondrial Membrane Potential Detection

BV-2 cells were treated with LPS (1 µg/mL, 24 h) ± 0.5 µM PK11195 (1 h pretreatment). The positive control group was pretreated with CCCP (C2006, Beyotime, Shanghai, China) at 37 °C for 30 min. After washing with PBS, the working solution (C2006, Beyotime, Shanghai, China) was added to all groups and incubated at 37 °C for 30 min, followed by two washes with the washing solution (C2006, Beyotime, Shanghai, China). Images were then captured using a fluorescence microscope (Olympus, Tokyo, Japan) and analyzed using Image-Pro Plus.

siRNA Preparation and Transfection

For the siRNA experiments, BV-2 cells (6 × 105 per well) were plated in six-well plates. After a 24-h incubation period, the cells were transfected with 100 nM siRNA (Genechem, Shanghai, China). The following siRNA sequences were designed to target ferredoxin 1 (FDX-1):

siRNAs were transfected into BV-2 cells using CALNP™ RNAi in vitro (DN001-05, D-Nano, Beijing, China), following the manufacturer’s instructions. After 24 h, the medium was replaced, and the cells were incubated in fresh medium for an additional 24 h. The levels of FDX-1 in the transfected BV-2 cells were then assessed using western blotting and qRT‒PCR.

Transmission Electron Microscope (TEM)

BV-2 microglial cell pellets were initially fixed in cold 3% glutaraldehyde phosphate buffer (pH 7.4) for 1 h and subsequently postfixed in 1% osmium tetroxide (OsO4). Following dehydration and embedding in EPON resin, ultrathin sections were prepared, stained with uranyl acetate and lead citrate, and analyzed using transmission electron microscopy (HT 7700-SS, HITACHI, Japan).

Statistical analyses

All data meeting the criteria for normal distribution are presented as the mean ± standard errors of the mean (SEM). Statistical analyses were conducted using GraphPad Prism 8.0 software. The Shapiro–Wilk test was used to assess the normality of the data. For comparisons between two groups with homogeneous variances, independent-samples t-tests were employed. In analyses involving three or more groups, one-way analysis of variance (ANOVA) was performed, followed by Bonferroni post-hoc correction. For data that did not adhere to a normal distribution, the Kruskal–Wallis H test was applied, followed by Dunn–Bonferroni t tests for multiple comparisons. The significance threshold was set at α = 0.05, and differences were deemed statistically significant when P < 0.05.