Animals and ICH model
The Institutional Animal Care and Use Committee at the Third Military Medical University approved this study (SCXK-PLA-20120011) and the procedures followed were in accordance with institutional guidelines. Two hundred and eighty-five adult male Sprague-Dawley rats (250–350 g) were used. ICH model establishment was refer to our previous studies.[15, 16] Briefly, nonanticoagulant whole blood was taken from the femoral artery of the rats. Then, the autologous blood was microinjected into the right caudate nucleus in 10 min. The coordinates is 0.2 mm anterior, 5.5 mm ventral, and 3.5 mm lateral to the bregma. The sham groups received only needle injection.
In the present study, the following three separate experiments were conducted:
To determine simvastatin-induced changes of LXA4 levels in plasma after ICH, 24 rats were randomly divided into three groups: sham, ICH + Veh (ICH + saline), ICH + Simva (ICH + simvastatin). The levels of LXA4 in plasm were assessed by Enzyme-Linked Immunosorbent Assays (ELISA) at 24hr and 72hr following blood injection, respectively (n = 4 per group, at each time point).
To validate the involvement of the LXA4/FPR2 pathway in simvastatin-mediated peripheral PMNs apoptosis promotion and subsequent neuroinflammation alleviation after ICH, we randomized 141 rats into the following five groups: Sham, ICH + Veh, ICH + LXA4 (an endogenous FPR2 agonist), ICH + Simva, ICH + Simva + Boc-2 (a selective FPR2 antagonist). Because our previous study has shown some corresponding data about the sham group, in present experiment, the sham group just undergo the measurements of PMNs count and proinflammatory factors protein level. Flow cytometric analysis of peripheral PMNs apoptosis (n = 6 per group), routine blood counts and immunofluorescence of MPO (n = 6 per group), Western blotting analysis of proinflammatory factors (n = 3 per group) were carried out 24hr after ICH induction. Brain water content measurement (n = 6 per group), Modified Neurological Severity Scores (mNSS, n = 6 per group) and corner test (n = 6 per group) were assessed at 1, 3 and/or 7 days after ICH.
P38 MAPK, the potential downstream signaling pathway of FPR2-mediated PMNs apoptosis, was evaluated in PMNs isolated from circulating blood of ICH rats. First, 12 rats were randomly divided into four groups: ICH + Veh, ICH + LXA4 (an endogenous FPR2 agonist), ICH + Simva, ICH + Simva + Boc-2 (a FPR2 selective antagonist). Western blotting analysis for p38, pp38, Mcl-1, Bax and Mcl-1/Bax ratio (n = 3 per group) were conducted at 24hr after ICH. Second, another 12 rats were randomly divided as the following four groups: ICH + Veh, ICH + SB203580 (p38 MAPK pathway inhibitor), ICH + Simva, ICH + Simva + P79350 (p38 MAPK pathway agonist). Western blotting analysis for Mcl-1, Bax and Mcl-1/Bax ratio (n = 3 per group) were conducted at 24hr after ICH.
Firstly, the simvastatin was prepared as a 4mg/ml solution according to our previous study.  This simvastatin stock was stored at −80°C and diluted with triple volume of sterile saline immediately before use. Animals received simvastatin injection (2 mg/kg/d, i.p) from five days before ICH until sacrificed.
Routine blood counts
Routine blood counts were conducted as previously described. First, 4 ml circulation blood were sampled from the heart of rat with the EDTA-anticoagulated tube. Then, give it a good shake. Next, 200 µl of sample was moved to an eppendorf tube and tested on the bench-top analyzer (Hemavet 950, Shandong excellent science instrument co. LTD., CHN).
As our previous described, the collected anticoagulated whole blood from rats was deal follow the instructions for density gradient centrifugation. Then, the PMNs cells were collected from the PMN-rich layer between Histopaque1083 and Histopaque1119. Next, the cells viability was examined with Trypan blue dye. The purity of isolated PMNs were tested with Wright-Giemsa staining. More than 95% of viability and purity were detected among these isolated PMNs.
PMNs apoptotic ratio detection
The apoptotic ratio of PMNs were detected refer to the previous method. The D-Hanks-washed cells were incubated on ice with 10 μl propidium iodide (PI) solution and 5 μl Annexin V-fluorescein isothiocyanate (FITC) solution for 15 min in the dark. Then, the apoptosis of PMNs were analyzed by the flow cytometry (BD LSRF Ortessa, USA).
Brain water content measurement
Brain water content was examined in rats 24 h and 72 h after surgery, as previously described.  Brains were removed under deep anesthesia, and a coronal tissue was sliced (4 mm thickness) around the injection needle tract. Brain sections were divided into four parts: ipsilateral basal ganglia, ipsilateral cortex, contralateral basal ganglia, and contralateral cortex. The cerebellum was the internal control. Brain sample weights were determined immediately after removal and then drying for 24 h in a 100 °C oven. The brain water content (%) was calculated as (wet weight-dry weight)/wet weight × 100 %.
Neurological function assessment
Neurological assessment was conducted with the modified Neurological Severity Score (mNSS) method and the corner test as previous.[17, 18] Briefly, the mNSS scale ranges from 0 to 18 (normal score, 0; maximal deficit score, 18). In corner test, each rat was leaded to a corner with 30° angle. Alternatively, the rat was allowed to turn left or right. After 10 times of repeated tests, the ratio of right turns was calculated by two blinded observers.
Cell counting was performed on brain sections. Three high power images were taken around the hematoma using a confocal microscope (LSM-780; Zeiss). MPO-positive cells were counted by two researchers in a blinded manner on 1, 3, and 7 days after ICH. The counts were performed on four consecutive brain sections.
Western blot analysis
Cell lysates were run on SDS/PAGE gels, and proteins were detected on nitrocellulose blots with enhanced chemiluminescence reagents (GE Healthcare). The following antibodies were used: TNF-α, diluted 1:500 (Beyotime, China); IL-6, diluted 1:500 (Beyotime, China); Mcl-1, p38 MAPK and phospho-p38 MAPK (1:1000, Cell Signaling, USA); Bax (1:500, Abcam, USA); complement C3 (1:500, Novus Biologicals, USA);α-Tubulin (1:1000, Boster Biological Technology, China); GAPDH (1:1000, bs-2188R; BIOSS, China). Membranes were blocked in 5% nonfat milk that was dissolved in Tris-buffered saline solution for 1 h, followed by overnight primary antibody incubation.
Brains were removed after fixative perfusion and cryoprotected in 30% sucrose in phosphate buffered saline (PBS). Serial sections were cut on a freezing microtome, blocked, and incubated in the primary antibodies: MPO (1:100, Abcam, USA). Then, the sections were incubated with secondary antibody: Alexa Fluor 555-conjugated goat anti-rabbit IgG (H+L) (1:300, Beyotime, China) at 37℃ for 3 hours. After washing, the sections were incubated with the appropriate fluorescent secondary antibody Alexa Fluor 555-conjugated goat anti-rabbit IgG (H+L) (1:300, Beyotime, China) and counterstained with DAPI.
In present study, the values are given as the mean±SD. Data were analyzed by one-way analysis of variance, followed by Scheffe’s post hoc test. Differences were considered statistically significant at a P-value < 0.05.