Animals and myocardial ischemia-reperfusion (IR) surgery

All experiments adhered to the guidelines set by the Animal Care and Use Committee of the National Institutes of Health (MD, USA). The study protocol was approved by the Ethics Committee of Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University (Nanjing, China). Male C57BL/6 mice aged 18–20 months were sourced from Aniphe Biolaboratory Inc. (Nanjing, China). Throughout the study, mice were group-housed and sustained in a controlled environment featuring a 12:12 light/dark cycle, temperature of 22 ± 1 °C, and 50 ± 10% humidity, with continuous access to food and water.

The myocardial IR procedure was adapted from the established method with slight changes (Curaj et al. 2015). In brief, mice were anesthetized using isoflurane and ventilated with a specialized animal ventilator (SAR-830/AP, CWE, Inc, PA, USA). A lateral incision was made between the fourth and fifth left ribs to expose the left ventricle. The left anterior descending artery (LAD) was then ligated with a 7 − 0 silk suture and released after 45 min. Visual confirmation of ischemia and reperfusion was achieved by observing heart muscle coloration and contractile variations through a stereomicroscope. Subsequently, the chest and skin were stitched separately.

Behavioral tests

24 h post-IR surgery, the mice underwent sequential behavioral assessments: open field test (OFT), elevated plus maze test (EPM), Y-maze test (YMT), and novel object recognition test (NORT). We maintained a gap of over an hour between each test to ensure reliable results. Using the automated video tracking system (XR-Xmaze, Shanghai Xinruan Information Technology Co., LTD, Shanghai, China), we tracked and analyzed each mouse’s behavior. To avoid interference from olfactory cues, chambers received thorough cleaning with 30% ethanol and were subsequently dried using paper towels ahead of each test.

Open field test (OFT)

For the OFT, mice were introduced to the center of an open field chamber and left to freely move for 5 min. We measured their total distance traveled to gauge their overall activity.

Elevated plus maze test (EPM)

The EPM served as a means to detect anxiety-related behaviors. Mice were delicately positioned at the maze’s intersection, oriented towards an open arm opposite the experimenter’s location. With the help of an overhead video camera, their activities were observed and logged for 5 min. We documented the duration spent in both the closed (CA) and open arms (OA), subsequently calculating the percentage of time they spent in the OA.

Y maze test (YMT)

YMT evaluated the mice’s short-term spatial memory. This three-armed maze, with each arm placed at a 120° angle relative to the others, allowed us to observe the mouse’s movement. When a mouse’s four limbs were inside an arm, it was considered a single entry. We documented the total arm entries and successful alternations, then computed the alternation score based on the formula: (number of successful alternations / (total arm entries − 2)) × 100.

Novel objective recognition test (NORT)

The NORT aimed to discern the recognition memory capacities of the mice. Before the actual test, mice were familiarized with the experimental setting 24 h in advance. The process was divided into two sessions: training and testing. During training, mice interacted with two identical objects in an open chamber for 10 min. The testing phase, conducted 24 h post-training, reintroduced the mice to the chamber, now containing one familiar and one new object, positioned as in the training. Using our video system, we monitored the exploration durations and derived the discrimination index by comparing the time spent with the novel object to the cumulative time spent with both objects.

Immunofluorescence staining (IF)

The mice were anesthetized by isoflurane and subsequently perfused with phosphate buffer saline (PBS) and phosphate-buffered 4% paraformaldehyde (PFA) via a transcardial method. Brains were immersed in 4% PFA for further post-fixation overnight, dehydrated with 30% sucrose, embedded in OCT, and stored at -80 °C. A frozen section machine (CM1950, Leica) cut the hippocampal sections into 30 μm slices coronally. Brain slices were washed with PBS for 3 times lasting 30 min followed by being blocked in 10% normal goat or donkey serum in TBST with 0.3% Triton X-100, and then primary antibodies in blocking buffer were applied overnight at 4 °C. The primary antibodies were used as follow: IBA1 (1:500, Rabbit, ab178846, Abcam), IBA1 (1:500, Goat, ab289874, Abcam), CD68 (1:500, Rat, BioLegend, 137,001), NLRP3 (1:500, Rabbit, ab270449, Abcam), PINK1 (1:200, Mouse, sc518052, Santa Cruz), Parkin (1:200, Mouse, sc32282, Santa Cruz), PSD95 (1:500, Mouse, ab13552, Abcam). The next day, slices were washed with PBS and incubated with the species-appropriate secondary antibodies. The secondary antibodies were all used as follows: Goat Anti-Rabbit Alexa Fluor 488 (1:1000, Ab150077, Abcam), Donkey Anti-Goat Alexa Fluor 647 (1:1000, Ab150135, Abcam), Goat Anti-Mouse Alexa Fluor 594 (1:1000, Abcam, Ab150113), Goat Anti-Rat Alexa Fluor 594 (1:1000, Abcam, Ab150160). Then, slices were washed with PBS and mounted with DAPI (1:1000, D8417, sigma) into PBS with 50% sterile glycerol. The images of the hippocampal CA1 region were captured by Olympus confocal microscope FV3000 with z-stack using a 40x oil objective. The 3D-reconstructed using the surface rendering function in Imaris 10.0.0. The CD68 or PSD95 inside IBA1 were filtered using the shortest distance to surfaces function, and the volume fraction was then quantified. The fluorescence intensity of NLRP3, PINK1, and Parkin in microglia was measured by Fiji software using the threshold method combined region of interest manager.

Western blot (WB)

The hippocampus was rapidly harvested after the mice were anesthetized by isoflurane. All procedures were performed on ice. Briefly, the hippocampus was homogenized in RIPA lysis buffer (50 mM Tris-HCl, 1% Triton X-100, 150 mM NaCl, 0.1% SDS) plus 0.5% Na3VO4, 10 mM NaF, 1 mM PMSF and protease inhibitor cocktail tablets, shake gently at 4 °C for 30 min, centrifuge at 10,000 g for 30 min at 4 °C, and carefully collect the supernatant. Protein concentration was determined by bicinchoninic acid (BCA) protein assay kit (23,225, Thermo Fisher Scientific, Waltham, MA, USA), and then, the samples were adjusted to an equal protein concentration with protein lysis buffer and 5× sample loading buffer. Samples were then boiled at 95 °C for 5 min and stored at -20 °C before the immunoblot analysis. Equal amounts of proteins in each group were added and separated by SDS/PAGE and transferred to the PVDF membrane. After that, the membrane was blocked by 5% non-fat milk in 1× Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. The incubation of the following primary antibodies was carried out overnight at 4 °C: NLRP3 (1:500, Rabbit, ab270449, Abcam), Caspase-1 (1:500, Rabbit, 24,232, CST), IL-1β (1:500, Mouse, 12,153, CST). PINK1 (1:500, Mouse, sc518052, Santa Cruz), Parkin (1:500, Mouse, sc32282, Santa Cruz), And then, the membrane were washed with TBST for 3 × 5 min, then incubated with species-appropriate HRP-conjugated secondary antibodies in 5% non-fat for 1 h at room temperature. After incubation, the membrane was washed with TBST for 3 × 5 min, and developed with enhanced chemiluminescence reagent. The protein expression level was quantified using Fiji software (1.54f, NIH, Bethesda, MD, USA).

Separation of hippocampal mitochondria, detection of mitochondrial membrane potential, and ATP assay

Tissue mitochondrial extraction kits (C3606, Beyotime, Shanghai, China) were applied for hippocampal mitochondrial separation according to the manufacturer’s instructions and used for further detection of mitochondrial membrane potential, and ATP assay. Mitochondrial membrane potential was evaluated by enhanced mitochondrial membrane potential assay kit with JC-1 staining kit (C2003S, Beyotime, Shanghai, China); the ATP assay was performed by the ATP Assay Kit (S0026, Beyotime, Shanghai, China) was used to measure the ATP levels of the hippocampus.

Golgi staining

The mice were anesthetized by isoflurane and the brains were rapidly harvested, washed with Milli-Q water, and immersed in buffers from FD Rapid GolgiStainTM Kit (FD Neurotechnologies, Inc., Columbia, Maryland) according to our previous report. Serial brain slices (110 μm) were cut by using a Vibratome (VT3000, Leica) and stained according to the manual of FD Rapid GolgiStainTM Kit. Bright-field microscopy (Olympus) with a 10 × objective at a magnification of 20× and 40× were taken the pyramidal neurons and dendritic morphology in the CA1 region by an observer blinded to the experiment, respectively.

For the hippocampal CA1 regions, apical dendrites on representative pyramidal neurons were sampled for the analysis. The tracings of neurons and quantification of dendritic total length, the number of branching points, and intersections at concentric circles at 50 μm intervals from the soma center were performed using Neuron J (version 1.4.3) and Sholl analysis (version v3.4.4, NIH, Bethesda, MD) of image J software plugins, respectively. The number of spines was counted by using FIJI software.

Electrophysiology recording

Mice were anesthetized using isoflurane and subsequently perfused transcardially with cold cutting solution, bubbled with 95% O2 and 5% CO2. This solution comprised (in mM): 93 NMDG, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, 2 thiourea, 5 Na-ascorbate, 3 Na-pyruvate, 0.5 CaCl2, and 10 MgSO4. Following perfusion, the brain was swiftly extracted, and transverse hippocampal slices of 400 μm thickness were prepared. These slices were incubated in the aforementioned solution, enriched with 12 mM NAC for 12 min at 30℃. Afterward, they were transferred to ACSF, bubbled with 95% O2 and 5% CO2, for an hour—half of this duration at 30℃ and the remainder at room temperature. The ACSF composition was (in mM): 92 NaCl, 2.5 KCl, 30 NaHCO3, 1.2 NaH2PO4, 25 glucose, 20 HEPES, 5 Na-ascorbate, 3 Na-pyruvate, 2 Thiourea, 2 CaCl2, and 2 MgCl2.

Recordings were conducted in a chamber with ACSF at 30 ± 1℃. Evoked fEPSPs were captured using a micropipette positioned within the hippocampal CA1 region’s stratum radiatum. A bipolar electrode stimulated the Schaffer-collaterals in the CA1. After setting a baseline for fEPSPs, the LTP protocol was initiated with 4 high-frequency stimulation trains. Post LTP induction, slope alterations were normalized to the average fEPSP slope observed during baseline, which was set at 100%. The LTP magnitude was determined by averaging responses recorded between 50 and 60 min post-HFS.

Statistical analysis

All analyses were conducted using GraphPad Prism 9.0 (version 9.4.0). Data sets underwent evaluation with one-way ANOVA or two-way ANOVA followed by Tukey’s post hoc test. Results are displayed as mean ± SEM. A P-value < 0.05 indicated statistical significance.