Animal and Treatment

C57BL/6J male mice aged 10 to 12 months were used in this study. Mice were housed in groups of 2 to 5 per cage under controlled environmental conditions, including a consistent room temperature of 25 °C and a 12-hour light/dark cycle. All mice had ad libitum access to food and water throughout the experimental period. All surgeries and procedures were conducted in accordance with established protocols of the Animal Care Committee of the First affiliated Hospital at Zhejiang University. To pharmacologically inhibit Caveolin-1 (Cav-1) expression, mice received intravenous injections of methyl-β-cyclodextrin (MβCD; 300 mg/kg) via the tail vein beginning one day prior to surgery. Injections were subsequently administered once every 24 h until the animals were euthanized for tissue collection and analysis.

Tibial Fracture Surgery

Anesthesia induction was achieved using 3% isoflurane, and maintained at 1.5% throughout the surgical procedure. Mice were positioned supine, and their limbs were secured to a heating pad using adhesive tape to maintain body temperature. After applying sterile drapes, the surgical site was prepared by shaving the fur around the knee joint and disinfecting the area three times with povidone-iodine solution. A horizontal skin incision was made at the level of the knee to expose the patellar tendon. A 10 ml syringe needle was inserted through the patellar tendon and advanced parallel to the tibial axis to create a channel in the tibial plateau. A 0.38 mm intramedullary pin was inserted through this channel and positioned within the tibial plateau. The tibia was subsequently fractured at the distal end, ensuring that the intramedullary pin remained in palce and spanned the fracture site to provide internal fixation. Hemostasis was achieved using a povidone-iodine swab, and the skin incision was closed using non-absorbable sutures. Mice were allowed to recover on a heating pad for 10 min post-surgery before being returned to their home cages. To ensure adequate postoperative analgesia, tramadol (30 mg/kg) was administered subcutaneously once daily until euthanasia.

Open-Field Test(OFT)

Prior to testing, the interior surfaces of the open-field chamber were thoroughly cleaned with medical-grade alcohol and ventilated to ensure a dry, scent-free environment. The appropriate parameters were configured in the Anymaze behavioral tracking software. Each mouse was gently removed from its home cage and placed in the center of the open-field arena. The chamber was then quietly closed, and the experimenter immediately left the testing area to minimize external disturbance. The Anymaze software automatically recorded the mouse’s movement trajectory and total distance traveled over a 5-minute session. After completion of the trial, each mouse was transferred to a designated holding cage. Once all animals in the group had been tested, they were returned to their original home cages. To eliminate olfactory cues and prevent behavioral bias, the chamber was cleaned with alcohol and wiped dry with absorbent paper between trials.

TraceFear Conditioning (TFC) Test

The TFC test is a widely used behavioral paradigm for evaluating associative learning and hippocampus-dependent memory in rodents [26]. In this experiment, the duration of freezing behavior serves as an index of fear memory retention, with shorter freezing times indicating greater memory impairment.

Training Phase

Before training, the TFC chamber was cleaned in the same manner as the OFT using medical alcohol and absorbent paper to eliminate olfactory cues. Each mouse was gently removed from its home cage and placed in the center of the chamber. The chamber door was closed quietly, and the experiment was initiated using Anymaze software. Mice were allowed to explore the chamber freely for 100 s. Following this habituation period, an auditory stimulus (80 dB tone) was delivered for 20 s, immediately followed by a 2-second foot shock (0.8 mA). After a 100-second inter-trial interval, this tone-shock pairing was repeated once more. Upon completion of the second cycle, mice remained in the chamber for an additional 60 s before being returned to a pre-designated holding cage. Mice in the surgery group underwent tibial fracture surgery approximately 10 min after the training session.

Test Phase

Three days after the training and surgery, mice were returned to the same chamber, but no tone or foot shock was administered during the test session. Freezing behavior was recorded continuously for 5 min using Anymaze software. The total freezing time was expressed as a percentage of the 5-minute test duration. A higher percentage of freezing time indicates better memory retention and cognitive performance, while a lower percentage suggests impaired fear memory and cognitive dysfunction.

Cell Culture and Treatments

Mouse brain microvascular endothelial cells (bEnd.3, Shanghai Zhong Qiao Xin Zhou Biotechnology) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, US) supplemented with 10% fetal bovine serum (FBS, Sigma, US) and 1% penicillin/streptomycinat under humidified 5% CO2 air environment at 37 ºC incubator. Cells were cultured in T25 flasks and passaged when they reached 80–90% confluence. After subculturing to the third passage, the cells were cultured in 6-well or 12-well plates for protein extraction or to prepare cell migration assays. Lipopolysaccharide (LPS, 1 µg/ml) was used to establish inflammation to the bEnd.3 at the designated timepoints. Cells treated with MβCD are required to be incubated with MβCD (5 mM, TargetMol, T4072) for 1 h after LPS stimulation.

Immunofluorescence (IF)

The mice brain tissue was first fixed overnight in 4% PFA and then dehydrated in 30% sucrose solution for at least 24 h, followed by embedding in OTC and freezing at -20 °C. Cultured cells were fixed in 4% paraformaldehyde (PFA) for 10 min and permeabilized and blocked in10% goat or donkey serum and 0.1% Triton-X for 1 h at the room temperature. Then the sample were probed with primary antibody including anti-Cav-1 (CST, 3267 S, 2.5 µg/mL), anti-CD31(R&D, AF3628, µg/mL), anti-claudin-5 (Thermo, 35-2500, 2.5 µg/mL), anti-MMP2 (abcam, ab92536, 8.4 µg/mL), and anti-MMP9 (abcam, ab76003, 9.3 µg/mL) at 4 °C for at least 16 h. Fluorescent secondary antibody including 594-Conjugated Goat Anti-Rabbit 488-Conjugated Donkey Anti-Goat (abcam, ab150129, 2 µg/mL), 594-Conjugated Goat Anti-Rabbit (proteintech, SA00013-4, 0.3 µg/mL), 488-Conjugated Goat Anti-Rat (abcam, ab150165, 1 µg/mL) was to bind the primary antibody in the dark at room temperature for 2 h. Finally, the samples were counterstained with DAPI (1 µg/ml) and imaged using the OLYMPUS microscope. Fluorescence images were quantified by Image J software. The quantification results of all fluorescence images were normalized to the average values of the 0 h group or 0 h + PBS group before statistical analysis.

BBB Permeability Assay

The surgical procedure for the mice was the same as previously described. Fifteen minutes prior to the scheduled tissue collection in mice, 10,000 MW Dextran Alexa Fluor (Thermo-Fisher, D3312) was administered via tail vein injection. After 15 min, the mice were euthanized with deeply anesthetized of isoflurane. Brain tissue was used for immunofluorescence staining. After endothelial marker CD31 staining, sections were observed under a 100x objective. Under normal conditions, the Dextran tracer (red) was co-labeled with the CD31 (green), indicating that the BBB remained intact, with no signs of leakage. If the Dextran tracer leaked outside the green blood vessels, it was considered an indication of BBB leakage.

Western-Blot

BEnd.3 or hippocampus were lysed in RIPA buffer, and the protein concentration was calculated by bicinchoninic acid assay (BCA). Proteins were isolated through 10% SDS‒PAGE and transferred to PVDF membranes. The membranes were blocked with 5% nonfat dry milk and then incubated overnight at 4 °C with primary antibodies, including anti-Cav-1(CST, 3267 S, 0.5 µg/mL), anti-ZO-1 (Thermo, 40-2200, 0.25 µg/mL), anti-claudin-5 (abclonal, A10207, 1 µg/mL) and anti-β-actin (proteintech, 66009-1, 0.25 µg/mL). The binding of primary antibodies was visualized with a goat anti rabbit HRP conjugated secondary antibody (ZSGB-Bio, ZB-2301, 1:4000) or goat anti-mouse HRP conjugated secondary antibody (ZSGB-BIO, ZB-2305, 1:4000).

ELISA

Cell culture and total protein extraction methods were as described above for cell culture and Western blot. The total protein was centrifuged at 4 °C, 2500 rpm for 20 min. The supernatant was collected and diluted 10-fold for the assay. ELISA kits for MMP2 (Fusheng Industrial, A106561) and MMP9 (Multi science, EK2M09) were used according to the manufacturer’s instructions. 100 µL of standard dilutions and diluted samples were added to the 96-well plate and incubated at 37 °C for 1 h. After washing the plate 5 times, 100 µL of enzyme-labeled antibody was added and incubated at 37 °C for 1 h. After washing the plate 5 times again, 100 µL of chromogenic substrate was added and incubated at 37 °C for 30 min. After adding the stop solution, the absorbance was measured at 450 nm and 630 nm using a microplate reader. A standard curve was established based on the dilutions of the standard samples and their absorbance, and the expression levels of each sample were calculated.

Statistics

All data were analyzed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA). All sets of continuous data were tested for normality using the Shapiro‒Wilk test, and fewer than 5% of the tests concluded that the set was nonnormal at the 0.05 significance level, confirming that the datasets met the assumption of a normal distribution. Two variants were compared by t test (two-tailed) for independent samples for the results, and comparisons between several groups were performed using ordinary one-way ANOVA. Data are presented as means ± standard error of the mean (SEM). Statistical significance was set at * P < 0.05, ** P < 0.005, *** P < 0.0005, **** P < 0.00005, and N.S indicates no significance.