The adult male C57BL/6 mice, about 8-weeks-old and weighing about 20–25 g each, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and were bred and maintained in the Animal Resource Centre of the Faculty of Tianjin Medical University. All study protocols were approved by the Animal Ethics Committee of Tianjin Huanhu Hospital and conducted in strict accordance with the ARRIVE Guidelines. In this study, these mice were randomly allocated to 3 groups: (1) Sham: Mice subjected to sham surgery only; (2) Mice subjected to ICH and intraperitoneally injected with MRS2578; (3) Mice subjected to ICH and injected with an equal volume of vehicle. For each, mice were evaluated for immunoblotting for selective markers, immunofluorescence, MRI and neurological defect. A total of 168 mice were used in this study, and 14 mice died during the experiment with a mortality rate of 8.3%.

The experimental ICH mouse model was induced using type IV collagenase (Sigma, St Louis, MO, USA), as previously described (Li et al. 2024; Yang et al. 2025). Briefly, the mice were anesthetized with an anesthetic containing 3% isoflurane and fixed in a prone position on the stereotaxic apparatus, keeping the head and body at the same level. Then, 0.0375U of collagenase was injected slowly to the right basal ganglia region (0.5 mm anterior and 2.3 mm lateral of the bregma, and 3.0 mm depth below the surface of the skull). After the injection, the needle remained stationary for 20 min. Then, the needle was slowly removed, and the skin was sutured. The temperature was maintained at 37.0 ± 0.5 °C during the experimental and early recovery periods.

The selective P2Y6 receptor inhibitor MRS2578 (# B2167) was purchased from ApexBio. The powder was dissolved in the mixture of DMSO, PEG300, Tween80 and ddH2O. And the storage concentration was 10 mg/ml. The ICH mice in MRS2578 group were injected intraperitoneally with MRS2578 (3 mg/kg) for 3 consecutive days, while the mice in ICH group were injected with the equal volume of DMSO solution diluted with PEG300, Tween80 and ddH2O.

The modified neurological severity score (mNSS) was performed to assess the global neurobehavioral defects on days 1, 3, 5, 7, and 14 after ICH. The test mainly includes 4 aspects including movement, sensation, balance and reflexes, with the score grade from 0 to 18. The higher the score, the more severe the neurological dysfunction.

Limb balance and coordination were assessed by the corner test on days 1, 3, 5, 7, and 14 after ICH. Briefly, we placed mice in a space with an angle of 30°, guiding them entering deep into the corner. When the whiskers were stimulated, which caused the mice to turn back to the open end. The number of right turns over ten trials was recorded. The results were calculated as right turn rate (number of right side turns/total number of turns × 100%).

Lesion volume and hematoma volume were quantified at day 3 after ICH using a 9.4-T small animal MRI, as previously described. T2-weighted image sequence (repetition time, 4500 ms; echo time, 65.5 ms; field of view, 28×28; image matrix, 256×256; slice thickness, 0.5 mm) was scanned to detect lesion volume and hematoma volume. The lesion and hematoma areas were manually outlined on each slice, then the areas in all slices were summed and multiplied by section thickness using image J. The lesion and hematoma volumes were measured by 2 investigators blinded to the tissues source.

On day 3 after ICH, mice were anesthetized and trans-cardially perfused with cold 0.9% saline. We selected cerebral tissues around the hematoma to extract total proteins using ice-cold RIPA buffer. Protein concentration was detected by a BCA Protein Assay Kits (Thermo Scientific). Proteins were electrophoresed and transferred onto a PVDF membrane, followed by blocking with 5% skim milk for 2 h. Then, membranes were incubated overnight at 4 °C with the following primary antibodies: P2Y6 receptor (1:2000, ApexBio), β-actin (1:10,000, SDN), TNF-α (1:200, abcam), and occludin (OCLN, 1:1000, Invitrogen), iNOS (1:1000, abcam), Anti-ZO-1 (1:2000, Invitrogen), NF-kB (1:1000, CST). On the next day, the membranes were washed and incubated with the species-appropriate horseradish peroxidase (HRP) labeled secondary antibody for 1 h at room temperature (RT). Then, the membranes were visualized using a Fusion FX VilBer LouRMAT System. Data analysis was performed by Image J.

On day 3 post-ICH, the mice were euthanatized and perfused for frozen brain section preparation. After fixing with 4% paraformaldehyde for 25 min and incubating with 3% bovine serum albumin (BSA) for 45 min, the 6 µm-thick sections were incubated overnight at 4 °C with the following primary antibodies: anti-P2Y6 receptor (1:2000, ApexBio), CD68 (1:2000, Bio-Rad), NeuN (1:2000, Thermo), Iba-1 (1:2000, Invitrogen), Anti-ZO-1 (1:2000,Invitrogen), Claudin-5 (CLN5, 1:1000, Invitrogen), TUNEL (In Situ Cell Death Detection Kit, Fliorescein). On the next day, the sections were washed and incubated with corresponding fluorescent secondary antibody in the dark at RT for 1 h. Nuclei were counterstained with DAPI (Abcam). Images were captured under a fluorescence microscope (Model BX-61; Olympus, Center Valley, PA).

Data are expressed as mean ± SEM. Comparisons between the two groups were made using the t-test to verify whether the differences were statistically significant. For comparisons between multiple groups, a one-way ANOVA was used to verify statistical significance between groups and then corrected for multiple analyses using GraphPad Prism 8.0.2 software. P < 0.05 is considered significant.