Animals and ethical statement
All procedures were approved by the ethics committee of Zhejiang University and followed the National Institutes of Health guidelines for the Care and Use of Laboratory Animals. Adult male C57BL/6J mice (age 8–10 weeks, weight 22–25 g,n = 341) were obtained from SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The mice were kept in a humidity-controlled room (25 ± 1 °C, 12-h light/dark cycle) and were raised with free access to food and water.
The autologous blood injection model of ICH was performed as previously described . Briefly, mice were anesthetized and maintained under 1% pentobarbital sodium, after which a volume of 25 µL of autologous blood was injected 2.5 mm to the right and 3 mm below the bregma at a 5° angle toward the midline. Mice in the sham group underwent the same procedure (anesthesia and needle insertion), except for injection.
All mice were randomly assigned to the following experiments (Fig. 1). A total of 341 mice including the dead ones, were used in this study.
Bexarotene is a highly selective RXR-α agonist approved by The Food and Drug Administration (FDA or USFDA) as an antineoplastic agent for the treatment of cutaneous T-cell lymphoma, with high blood-brain barrier permeability and a good safety profile . To assess the effects of RXR-α activation on hematoma clearance and neurological function after ICH, the RXR-α agonist bexarotene was used. Neurological function (as assessed by the cylinder test, corner turn test, and forelimb placement test) was tested after ICH. Mice were assigned into three groups: sham group, ICH + vehicle group (10% Dimethyl sulfoxide (DMSO), in saline), and ICH + bexarotene group (5 mg/kg) (n = 9). The sham group received the same volume of vehicle intraperitoneally at the same time points after ICH induction. A T2* weighted magnetic resonance imaging (MRI) scan was used to measure the hematoma volume at 1, 3, 7, 14, and 28 days after ICH. Mice were randomly divided into two groups: ICH + vehicle (10% DMSO, in saline), and ICH + bexarotene (5 mg/kg) (n = 6).
To assess the expression pattern of RXR-α and PPAR-γ after ICH, mice were assigned to five groups: a sham, ICH 1 d, ICH 3 d, ICH 7 d, and ICH 14 d group (n = 6). Whole-cell lysates Western blots(n = 6) and cytoplasmic and nuclear protein Western blots (n = 6) were performed at different time-points. Next, the cellular location of RXR-α was assessed by double immunofluorescence staining in ICH (3 days) group (n = 6). To assess the nuclear translocation of RXR-α, mice were divided into three groups: a sham group, ICH + vehicle group (10% DMSO, in saline), and ICH + bexarotene group (n = 6). Whole-cell lysate Western blots (n = 6) and cytoplasmic and nuclear protein Western blots (n = 6) in these groups were performed at 3 days after ICH. To determine whether PPAR-γ plays a role in RXR-α activation after ICH, the RXR-α agonist bexarotene and PPAR-γ antagonist GW9662 were used. To assess the nuclear translocation of PPAR-γ, mice were divided into four groups: a sham group, ICH + vehicle group (10% DMSO, in saline), ICH + bexarotene + vehicle group, and an ICH + bexarotene + GW9662(4 mg/kg) (n = 6). Whole-cell lysate Western blots (n = 6) and cytoplasmic and nuclear protein Western blots (n = 6) in these groups were performed at 3 days after ICH.
To assess the mechanism underlying the role of RXR-α activation in ICH, mice were randomly assigned to four groups: a sham group, ICH + vehicle group (10% DMSO, in saline), ICH + bexarotene group + vehicle group (10% DMSO, in saline), and an ICH + bexarotene (5 mg/kg) + GW9662 (4 mg/kg) group. Immunofluorescence staining (n = 4), Western blots (n = 6), and enzyme-linked immunosorbent assays (ELISAs) (n = 6) were used to assess microglia/macrophages polarization.
Next, we assessed whether PPAR-γ plays a role in the protective effects linked to RXR-α activation. To this end, the cylinder test, corner turn test, and forelimb placement test were used after ICH. Mice were randomly divided into three groups: an ICH + vehicle group (10% DMSO, in saline), ICH + bexarotene + vehicle group (5 mg/kg), ICH + bexarotene (5 mg/kg) + GW9662 (4 mg/kg) (n = 6). The sham group received the same volume of vehicle intraperitoneally at the same time points after ICH induction. An MRI scan was used to measure the hematoma volume at 1, 3, 7, and 14 days after ICH. Mice were randomly divided into three groups: an ICH + vehicle group (10% DMSO, in saline), ICH + bexarotene + vehicle group (5 mg/kg), and ICH + bexarotene (5 mg/kg) + GW9662 (4 mg/kg) group (n = 6).
Bexarotene (MedChem Express, New Jersey, USA) was dissolved in 10% dimethyl sulfoxide (DMSO) as previously described. The selective PPAR-γ antagonist GW9662 (MedChem Express, New Jersey, USA) was diluted in 10% DMSO. bexarotene solution (5 mg/kg) or an equal volume of vehicle or bexarotene solution (5 mg/kg) + GW9662 solution (4 mg/kg) was administered intraperitoneally 1 h after ICH for the first time, followed by daily injections until sacrifice. The dosage and time points of bexarotene and GW9662 were based on a previous study[21, 22].
Calculation of hematoma volume
Mice were anesthetized with 1% pentobarbital sodium for the MRI examination. An MRI was performed at days 1,3,7,14 and 28 after ICH in a 3.0-T MRI scanner. The MRI included a T2* sequence. The scanning parameters for T2* weighted imaging were: TR/TE = 2200/103.8 ms, Number of averages = 10, acquisition matrix = 208 × 208, voxel size = 0.12 × 0.12 × 1 mm, flip angle = 130°, slices = 5. MRI image datasets were obtained in the Digital Imaging and Communications in Medicine (DICOM) format. The data were transformed into the NIfTI (.nii) format and then assessed with 3D Slicer. The 3D-Slicer method is one of the software methods serving to measure the volume of a hematoma(http://www.slicer.org/). Hematomas were manually identified pixel by pixel in each slice. Next, a 3D model was established and the hematoma volume was calculated by adding up the volume of the pixels.
Neurobehavioral functions were evaluated by a forelimb placing test, forelimb use asymmetry (cylinder) test, and a corner turn test, as previously reported . Baseline data were recorded for the reduction of variability and identification of the preferential side. The neurological scores were evaluated by a blinded observer.
Forelimb placement can be assessed by stimulating the mouse's vibrissae to trigger a response. To test the function of the forelimbs, the researchers held the animal's torso and hung the forelimbs freely, while brushing its vibrissae on the corner edge of a table. Non-brain-damaged animals usually respond to placing the forelimb on the table on the same side as the affected side, while ICH mice will be impaired in the placement of their paws. This test can be scored by counting the percentage of placements.
For the forelimb use asymmetry test (cylinder test), the mouse is placed in a transparent cylinder and observed the independent wall contacts. The behavior score was recorded as the number of times the ipsilateral (unimpaired) forelimb (I), contralateral (impaired) forelimb (C), and both forelimbs (B). A single overall limb use asymmetry score was calculated as follows: Limb use asymmetry score=[I/(I + C + B)]-[C/(I + C + B)].
For the corner test, a mouse was then placed between the boards facing the 30º angle corner. As the mouse approaches the corner, both sides of the vibrissae were simultaneously stimulated causing the animal to rear and turn 180º, the number of the right turns was then calculated.
Immunofluorescence double labeling
Coronal sections were blocked with 5% Bovine serum albumin (BSA) and 0.3% Triton X-100 and then incubated with primary antibodies overnight at 4 °C. The primary antibodies used were mouse anti–NeuN (1:500, ab-104224, Abcam, MA, USA), goat anti–Iba-1 (1:500, ab-5076, Abcam, MA, USA), mouse anti–GFAP protein (1:500, ab10062, Abcam, MA, USA), rabbit anti-RXR-alpha antibody (1:250, ab125001, Abcam, MA, USA), rabbit anti-Arg1 (1:500, 16001-1-AP, Proteintech, Hubei, China), rabbit anti-Nitric oxide synthase (iNOS) Antibody (18985-1-AP,1:500, Proteintech, Hubei, China), and rabbit anti-PPAR-γ antibody (1:250, ab178860, Abcam, MA, USA). The sections were incubated with secondary antibodies at room temperature for 2 h. Finally, the sections were observed and analyzed using a fluorescence microscope (Olympus, Tokyo, Japan). Photomicrographs were saved and merged using the Image-Pro Plus software.
Enzyme-linked immunosorbent assay
Brain samples were homogenized in chilled lysis buffer containing protease and phosphatase inhibitor cocktails (P1005, Beyotime, Shanghai, China). ELISA kits for mouse TNF-α (Boster, EK0527, Wuhan, China) were used to assess the levels of TNF-α in the brain. The total protein content of each sample was determined by a bicinchoninic acid (BCA) assay (ThermoFisher, Waltham, MA USA). An equal amount of protein brain homogenates were diluted at a 1:10 ratio with the sample diluent provided with the kits, and all procedures were performed per the manufacturer’s instructions.
Western blot analysis
A Western blot analysis was performed as previously described . Briefly, the basal ganglia were homogenized and centrifuged for 15 min (13,000 g, 4 °C). For the whole cell lysates, tissue proteins from basal ganglia were lysed using RIPA lysis buffer. For the extraction of cytoplasmic and nuclear protein, the cytoplasmic and nuclear proteins were extracted using Cytoplasmic and Nuclear Protein Extraction Kit ( P0027, Beyotime, Shanghai, China). Proteins were assessed using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA USA). An equal amount of protein (40 µg) was suspended in a loading buffer (denatured at 95 °C for 5 min), loaded on an SDS-PAGE, and transferred to nitrocellulose membranes. Next, the membranes were blocked with a nonfat dry milk buffer for 1 h and incubated overnight with the primary antibody. The membranes were then incubated with the secondary antibody for 1 h at room temperature. Bands were visualized using the ECL Plus chemiluminescence reagent kit (Amersham Bioscience, Arlington Heights, IL). The band densities were quantified using Image J software. The primary antibodies used in this study were rabbit anti-PPAR-γ (1:1000, Abcam, Cambridge, UK, ab45036), rabbit anti-RXR-α antibody (1:1000, ab125001, Abcam, MA, USA), rabbit anti-Arg1 (1:1000, 16001-1-AP, Proteintech, Hubei, China), mouse anti-GAPDH (1:5000, ab8245, Abcam, Cambridge, UK), mouse anti-β actin (1:5000, ab8227, Abcam, Cambridge, UK), and rabbit anti-histone H3 (1:2000, #9715, Cell Signaling Technology, MA, USA).
All data in this study are expressed as the mean ± standard error of the mean. For data that meet a normal distribution and homogeneity of variance, differences among the groups were analyzed using a one-way analysis of variance (ANOVA) followed by a Tukey’s multiple comparison test. A Kruskal-Wallis test with a Bonferroni correction was used for the non-normally distributed data. All statistical analyses were performed in SPSS (version 22.0). A p-value < 0.05 was considered statistically significant.