Excluding 6 rats that died of an overdose of anesthetic before modeling, total 213 adult male Sprague-Dawley rats (250–300 g) were used for this study. Rats were housed under specific pathogen-free conditions and had free access to food and water. Animals were sacrificed at the endpoint under deep anesthesia using an overdose of intraperitoneal pentobarbital. All efforts were made to minimize suffering and animal numbers according to the Guide for the Care and Use of Laboratory Animals; the study was approved by the Animal Care and Use Committee at the Third Military Medical University.

To induce ICH, rats were anesthetized with an intraperitoneal injection of 5% chloral hydrate (350 mg/kg). A feedback-controlled heating pad was used to maintain body temperature at 37.0°C. A cranial burr hole (about 1 mm) was drilled, and a 29-gauge needle was inserted stereotaxically into the right basal ganglia (coordinates: 0.2 mm anterior, 5.5 mm ventral, and 4.0 mm lateral to the bregma) (19, 20). A total of 100 µl autologous arterial blood was infused at a rate of 10 µl/min using a microinfusion pump in each rat. The sham groups received only a needle injection into the right basal ganglia.

Brain water content was examined 24 and 72 h after surgery. As previously described (21), animals were anesthetized with an intraperitoneal injection of 5% chloral hydrate (350 mg/kg). Brains were removed, and the tissue was sliced coronally (4 mm thickness) around the hematoma. Samples were divided into four parts: ipsilateral basal ganglia, ipsilateral cortex, contralateral basal ganglia, and contralateral cortex. Cerebellum was regarded as the internal control. Sample weights were determined immediately after removal and after drying for 24 h in a 100°C oven using an electric analytical balance. Brain water content (%) was calculated as (wet weight − dry weight)/wet weight × 100%.

Behavioral outcomes were assessed in a blinded fashion at 24 and 72 h after surgery. The neurological abnormalities were assessed by the mNSS method. The evaluation was performed by an investigator blinded to the experimental scheme. The mNSS is a composite test of motor, sensory, and balance functions. Neurological function was graded on a scale of 0–18 (normal score, 0; maximal deficit score, 18) (22). For the forelimb placing test, each rat was tested 10 times for each forelimb; the percentage of trials that the rat placed the appropriate forelimb at the edge of the countertop in response to the vibrissae stimulation was determined. Testers were experienced and blind to the condition of the animal. The mean neurological score was evaluated by two blinded observers (23).

Fluoro-Jade C staining was used to assess neuronal degeneration. Briefly, sections were rinsed for 5 min in basic alcohol, followed by a 2-min rinse in 70% alcohol. Then, the sections were rinsed in distilled water and incubated in 0.06% KMnO₄ for 10 min. They were rinsed in distilled water to remove excess KMnO₄ and incubated in 0.0001% Fluoro-Jade C stain (Millipore, Boston, MA, USA) stain in 0.1% acetic acid for 10 min. Following Fluoro-Jade C labeling, the sections were rinsed three times in distilled water, air dried for 10 min, cleared in xylene, and covered with DPX. TUNEL staining was performed using an in situ cell death detection kit-POD (Roche, Switzerland) to reveal DNA damage according to the manufacturer’s instruction (24). High-power images (×40 magnification) were taken around the hematoma using a digital camera. Fluoro-Jade C and TUNEL-positive cells were counted. Counts were performed on four areas in each brain section.

PCR was performed and analyzed as previously described (25). Total RNA from brain tissue around the hematoma was extracted using Qiagen RNeasy mini kits. One microgram of RNA was reverse-transcripted and cDNA was synthesized using the PrimescriptTM RT kit (Takara, Dalian, China), substituting DNase and RNase-free water for no-RT controls. qPCR reactions were set up in 25 µl using SYBR Premix Ex TaqII kit (Takara, Dalian, China) and conducted on a CFX-96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The running procedure was 30 s at 95°C, 40 cycles of 5 s at 95°C, and 30 s at 60°C, following a melt curve. The qPCR primers were listed in Table S1 in Supplementary Material. Gene expression was quantified with standard samples and normalized with GAPDH. The data are expressed as normalized messenger RNA (mRNA) expression (fold mRNA increase).

Immunofluorescence staining was performed as previously described (21). Briefly, free-floating slices were incubated with primary goat anti-Iba1 (1:200, Abcam, Cambridge, United Kingdom) at 4°C overnight, followed by Alexa 555-labeled rabbit anti-goat IgG (H + L) (1:500; Beyotime, Wuhan, China) secondary antibody (1 h, 37°C). Sections were washed and blocked with 10% normal goat serum for 1 h, then incubated overnight with mouse anti-CD68 (1:200, 1:500, AbD Serotec, Oxford, UK) or rabbit anti-CD206 (1:400, Santa Cruz Biotechnologies, Dallas, TX, USA). Finally, sections were incubated with the appropriate secondary antibodies for 1 h at 37°C. Colocalization was examined using a fluorescent microscope (Zeiss, LSM780).

Western blot assays were performed as described previously (26). A total of 50 µg of prepared protein was loaded into each lane of SDS-PAGE gels. Gel electrophoresis was performed, and protein was transferred to a nitrocellulose membrane. The membrane was blocked in Carnation^® non-fat milk and probed with primary and secondary antibodies. The following primary antibodies were used: rabbit anti-CB2R (1:500, Abcam, Cambridge, UK), mouse anti-CD68 (1:500, AbD Serotec, Oxford, UK), rabbit anti-CD206 (1:500, Santa Cruz Biotechnologies, Dallas, TX, USA), rabbit anti-phospho-CREB (Ser133; 1:1,000, Cell Signaling Technology, Boston, MA, USA), rabbit anti-phospho-PKAC (Thr197; 1:1,000, Cell Signaling Technology, Boston, MA, USA), rabbit anti-β-tubulin (1:1,000, Abcam, Cambridge, UK), and mouse anti-β-actin (1:1,000, Santa Cruz Biotechnologies, Dallas, TX, USA). Then, membranes were incubated in the appropriate HRP-conjugated secondary antibody (diluted 1:1,000 in secondary antibody dilution buffer) for 1 h at 37°C. Protein bands were visualized using a nickel-intensified DAB solution, and the densitometric values were analyzed using Image J software. The housekeeping proteins β-tubulin and β-actin were used as internal controls.

Intracerebroventricular infusion was performed as described previously (27). Rats were anesthetized with an intraperitoneal injection of 5% chloral hydrate (350 mg/kg). The needle of a 10-µl Hamilton syringe (Microliter No. 701; Hamilton Company) was inserted through a burr hole in the skull into the left lateral ventricle, according to the following coordinates: 1.5 mm posterior, 4.2 mm ventral, and 0.8 mm lateral to the bregma. CREB-1 siRNA or an irrelevant scrambled siRNA [500 pmol each in 1 µl siRNA dilution buffer (Santa Cruz Biotechnology)] was injected (0.5 µl/min) using a microinfusion pump at 24 h before ICH induction. The needle was removed 10 min later to prevent backflow. The burr hole was sealed with bone wax, and skin incisions were closed with sutures after the needle was removed. All rats received JWH133 (1.5 mg/kg) intraperitoneally 1 h after ICH. CREB-1 siRNA consists of three different siRNA duplexes to improve the knockdown efficiency. All CREB-1 siRNA sequences were provided in 5′–3′ orientation as shown in Table S2 in Supplementary Material.

Data are reported as the mean ± SD. Data were analyzed using one-way analysis of variance tests followed by Student–Newman–Keuls tests. A non-parametric test (Kruskal–Wallis H) was used if the data were not normally distributed, followed by a Nemenyi test when a two-group comparison was necessary. Differences were considered statistically significant at P < 0.05.

We first investigated whether CB2R alterations would respond to brain injury after ICH. Data showed that CB2R levels around hematoma (Figure 1A) were significantly increased at 24 h after ICH when compared with the ICH 0 h group (P < 0.05) and remained at a high level until 72 h (Figures 1B,C).

To assess brain edema and neurobehavioral outcomes after ICH, brain water content and behavioral testings, including mNSS test and forelimb placing test, were used. Data showed that both at 24 and 72 h after surgery, rats subjected to ICH had increased perihematomal edema in the ipsilateral basal ganglia (P < 0.01 vs. Sham; Figures 2A,B). The animals also had significantly worse mNSS test performance (P < 0.01 vs. Sham; Figures 3A,B) and forelimb placing scores (P < 0.01 vs. Sham; Figures 3C,D). In ICH + JWH groups, the perihematomal brain edemas were significantly reduced (P < 0.05 vs. ICH + Vehi; Figures 2A,B), and the neurobehavioral outcomes were significantly improved (P < 0.05 vs. ICH + vehi; Figures 3A–D). All of these JWH133 effects were reversed by SR144528.

To investigate the neuronal degeneration after ICH, Fluoro-Jade C and TUNEL staining were used. At 24 h after ICH, the treatment of JWH133 significantly decreased the number of Fluoro-Jade C-positive cells (497 vs. 720/mm² in ICH + Vehi group, P < 0.01) (Figures 4A,B). Similarly, the number of TUNEL-positive cells were significantly decreased in the ICH + JWH group (524 vs. 325/mm² in ICH + Vehi group, P < 0.01) (Figures 4A,C). The result indicated that JWH133 could significantly attenuate neuronal death and DNA damage after ICH.

To investigate the characteristics of microglial activation during the acute phase following ICH induction, gene levels of M1/M2 markers were investigated time dependently. After ICH, M1-associated markers CD32, CD68, and CD86 increased immediately and peaked at 6 h, and remained a high level to 72 h (Figures 5A,C,E). M2-associated markers Arg-1, Ym-1, and CCL-22 increased slowly and peaked at 24 h, and kept a low level from 6 to 72 h (Figures 5B,D,F). However, the use of JWH133 significantly inhibited the increase of M1-associated mRNA levels (Figures 5A,C,E) and promoted M2-associated mRNA levels (Figures 5B,D,F), with the most obvious differences at 24 h, and all the effects could be significantly reversed by SR144528 (Figures 5A–E). Consistent with the qPCR results, immunofluorescent double-labeled staining and western blot results showed that the M1-associated marker CD68 was also more significantly upregulated than M2-associated marker CD206 at 24 h after ICH (Figures 6A,B). Treatment with JWH133 promoted the protein expression of CD206 and inhibited CD68 expression (Figures 6A–C). These results indicated that CB2R stimulation could drive the acquisition of M2 polarization in microglia and reduce M1 phenotype.

Inflammatory cytokine secretion plays an important role in brain injury after ICH. In this study, the mRNA expression levels of both pro-inflammatory (IL-1β, TNF-α, and iNOS) and anti-inflammatory cytokines (IL-4, IL-10, and TGF-β) in perihematomal brain tissue were detected at different time points after ICH. Expression levels of pro-inflammatory cytokines increased immediately and IL-1β peaked at 6 h (Figure 7A), while TNF-α and iNOS (Figures 7C,E) peaked at 24 h post-ICH. The gene levels of IL-4, IL-10, and TGF-β were upregulated at 6 h after ICH and reached a peak at 24 h (Figures 7B,D,F). However, treatment with JWH133 prevented the increase of IL-1β, TNF-α, and iNOS at 6 h after ICH induction, and the effect persisted to 72 h, with the most significant changes occurring at 24 and 72 h (Figures 7A,C,E). In contrast, IL-4, IL-10, and TGF-β were significantly increased at 6 h, with the most significant changes occurring at 24 h (Figures 7B,D,F). All of the JWH133-mediated effects were prevented by treatment with SR144528.

To further explore the underlying mechanisms, we tested the expression of pPKA–CREB signaling pathway. Results of western blot showed that pPKA and pCREB expression was significantly lower in ICH + Vehi group when compared with Sham group (P < 0.01), and the ICH + JWH group had significantly higher expression of pPKA (P < 0.01) and pCREB (P < 0.05) when compared with the ICH + Vehi group (Figures 8A,B), indicating that pPKA–CREB signaling pathway was significantly upregulated by JWH133 treatment. The pretreatment of SR144528 prevented the upregulation effects.

cAMP-response element binding protein (CREB) in vivo knockdown was performed to investigate the potential role of CREB in the effects of JWH133 induced the increase of microglial M2 polarization and the decrease of M1 phenotype. Pretreatment with CREB-1 siRNA sufficiently downregulated the expression of CREB both at gene and protein levels (Figures S1A,B in Supplementary Material). Data showed that CREB-1 siRNA abolished the JWH133-induced increase of M2-associated markers CD206 and Ym-1 at protein or gene level (Figures 9B,D,F), as well as the decrease of M1-associated markers CD68 and CD32 in protein or gene levels (Figures 9A,C,E). These results demonstrated that PKA/CREB signaling pathway plays a crucial role in the JWH133-mediated acquisition of M2 phenotype of microglia.