Experimental design
The following four separate experiments and the summary of experimental groups are depicted in Figure S1. Histological images and functional outcomes were assessed in a blinded manner. The investigators were blinded for the surgical procedures and treatment.
Experiment 1: Time course of endogenous CCL17 and CCR4 and their cellular localization after SAH.
In this section, 24 rats were randomly assigned to 6 groups: sham, SAH-3 h, SAH-6 h, SAH-12 h, SAH-24 h, and SAH-72 h. The expression of endogenous CCL17 and CCR4 after SAH was detected via western blot at every time point (n = 3 for each group). An additional rats were used for immunofluorescence (IF) staining to localize CCL17 and CCR4 in sham and SAH-24 groups (n = 3 for each group). CCL17 secretion from neurons was proven by western blot and ELISA assay in vitro. The degree of CCR4 expression was assessed in rat primary neurons, astrocytes, and microglia in vitro.
Experiment 2: The effects of rCCL17 treatment on EBI after SAH.
To determine the appropriate treatment dosage for rCCL17, a total of 95 rats were randomly assigned to 5 groups for neurobehavior evaluation: sham, SAH+vehicle, SAH+rCCL17 (10 μg/kg), SAH+rCCL17 (30 μg/kg), and SAH+rCCL17 (60 μg/kg). The rCCL17 was administered intranasally (i.n.) at 1 h following SAH. Neurobehavioral functions (Modified Garcia Scores and Forelimb Placement Tests) and brain water content (BWC) were evaluated at 24 h and 72 h after SAH (samples were shared, n = 6/group). Immunofluorescence staining (n = 3/group) and qPCR (n = 4/group) were performed. To explore the efficacy of the function of rCCL17 on EBI and microglial morphology was detected at 24 h after SAH.
Experiment 3: The role of the CCR4/mTORC2 signaling pathway post-SAH.
To examine the role of rCCL17 and CCR4 following SAH, 102 rats were randomly divided into 6 groups: sham, SAH+vehicle, SAH+rCCL17 (60 μg/kg), SAH+DMSO, SAH+AZD2098 (1 mg/kg), and SAH+JR-AB2-011 (1 mg/kg). The specific CCR4 inhibitor, AZD2098, and selective mTORC2 inhibitor, JR-AB2-011, were given via intraperitoneal (i.p.) administration 1 h prior to SAH induction. Samples were collected 24 h post-SAH. Changes in mTORC2 and ERK signaling were measured via western blot analysis (n = 3 per group) and the M2-like microglia biomarkers were examined by qPCR (n = 4 per group). Neurobehavioral functions and BWC were evaluated at 24 h and 72 h after SAH (samples were shared, n = 5/group/time point). Immunofluorescence staining (n = 3/group) and qPCR (n = 4/group) were performed. To explore the efficacy of rCCL17 on EBI and microglial morphology, TUNEL, FJC staining, and IF were performed at 24 h after SAH (n = 3 per group).
Experiment 4: The role of CCL17/CCR4/mTORC2 axis in microglia post-SAH.
In this experiment, we investigated the effects of rCCL17-regulated microglia after SAH. A total of 102 rats were randomly divided into 6 groups: sham+AAV-CD68-Control, SAH+vehicle+AAV-CD68-Control, SAH+rCCL17+AAV-CD68-Control, sham+AAV-CD68-shRictor, SAH+vehicle+AAV-CD68-shRictor, and SAH+rCCL17+AAV-CD68-shRictor. Adeno-associated virus was administered via intracerebroventricular injection 21 days before SAH induction. Samples were collected 24 h post-SAH. The changes in mTORC2 and ERK signaling were measured by western blot analysis (n = 3 per group) and the M2-like microglia biomarkers were examined by qPCR (n = 4 per group). Neurobehavioral functions and BWC were evaluated at 24 h and 72 h after SAH (samples were shared, n = 5/group/time point). Immunofluorescence staining (n = 3/group) and qPCR (n = 4/group) were performed. To explore the efficacy of rCCL17 on EBI and microglial morphology, TUNEL, FJC staining, and IF were performed at 24 h after SAH (n = 3 per group). The regulatory function of CCL17 on M2-like polarization of microglia was demonstrated in rat primary microglia in vitro.
Animals and Drug administration
Adult male Sprague-Dawley rats (10-week-old, 300-320g) were housed in a temperature- and humidity-controlled environment with ad libitum access to food and water. The light in the room was controlled in a 12-h light/dark cycle. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Zhejiang University and in accordance with the National Institutes of Health guide for the care and use of laboratory animals.
AZD2098 and JR-AB2-011 were dissolved in 10% DMSO and administered via i.p. injection. The rCCL17 was dissolved in sterile deionized water and administered via i.n. administration. Three different dosages of rCCL17 (10, 30, and 60 μg/kg) were given to rats at 1 h after SAH.
SAH Model and SAH Grading
The endovascular perforation SAH model was conducted as previously described 21. Briefly, isoflurane-anesthetized rats were intubated with mechanical ventilation. A sharp 4–0 monofilament was inserted from the left external carotid artery to the internal carotid artery and perforated the bifurcation of the anterior and middle cerebral arteries. Rats in the sham group underwent the same procedures without vessel puncture. Measurements of respiration, heart rate, skin pigmentation, and pedal reflex were recorded intraoperatively every 5 min to confirm the anesthetic status and prevent distress.
The SAH grading was blindly evaluated by two independent investigators according to the previous study 22. Briefly, the basal cistern was divided into six segments and scored from 0 to 3 based on the subarachnoid blood clot. The final score combined all segments and ranged from 0 to 18. If there was no blood present, a score of 0 was assigned. Rats with a score under 9 were excluded from this study.
Primary hippocampal neuron, microglia and astrocytes cultures
Within two days after birth, newborn Wistar rat pups were anesthetized and decapitated. The brain was quick removed, soaked in Hank’s solution, and stored at 4 ℃. The hippocampus was dissected rapidly under a microscope ice bath, and the tissue was cleaned three times with 5mL refrigerated DMEM/F12 medium. The hippocampal tissue was digested with 2 mL pectinase and gently agitated twice. The cell suspension was removed to repeat the process with pectinase until complete digestion was achieved. After digestion, the cells were evenly mixed with the culture medium from hippocampal neurons and counted for lamination. Half-volume of medium was replaced every other day 24 h, the neuronal state was observed for 9 days and subsequent experiments were carried out.
Simultaneously, cerebral cortices were enzymatically and mechanically dissociated and cells were seeded in DMEM/F12 medium supplemented with 10% FBS, 1% penicillin-streptomycin, 1 mM sodium pyruvate. The medium was replaced every 4 days. On day 14, the medium was removed, and cultures were trypsinized to isolate the monolayer of astrocytes and adherent microglia.
Oxygenated hemoglobin (OxyHb, 10 μM, Sigma-Aldrich, USA) was introduced into the medium for 24 h to simulate SAH insult in vitro as previously described 23.
Elisa assay for CCL17
The rat primary neuron was treated with or without OxyHb (10 μM, Sigma-Aldrich, USA) to simulate SAH for 24 h. The supernatant was collected for quantitative analysis of endogenous CCL17 using ELISA kit (LifeSpan BioSciences, LS-F4911) following the manufacturer’s instruction.
shRictor adeno-associated virus vector construction
To knockdown the expression of Rictor (a composition of mTORC2) in microglia in vivo, we constructed adeno-associated virus (AAV)-CD68 mediated Rictor interference virus vector pHBAAV2/9-CD68-shRictor (AAV-CD68-shRictor) (Hanbio Technology). The sequences were GGCTGTGATATTCTAAAGT (sense) and ACTTTAGAATATCACAGCC (anti-sense). The verification of the inhibitory effects was conducted 3 weeks later after stereotaxic injection to the cerebral ventricle.
Modified Garcia Score and Beam balance test
The modified Garcia Score and Beam balance test were used to assess short-term neurological deficits at 24 h and 72 h post-SAH as previously described24. Briefly, the modified Garcia test with a 21-point score was conducted to evaluate spontaneous activity, axial sensation, symmetry of limb movement, vibrissae proprioception, and forelimb walking. Beam balance test is used to assess the rat’s ability to walk on the wooden beam for 1 min. The evaluation was as follows: 0 = not walk and fall; 1 = not walk but remain on beam; 2 = walk but fall; 3 = walk < 20 cm; and 4 = walk beyond 20 cm.
Brain water content measurement
The wet/dry method was used to measure BWC according to previous study25. The rats were sacrificed and the brains were collected immediately at 24 h after surgery and cut into four sections: left cerebral hemisphere, right cerebral hemisphere, cerebellum and brain stem. Each section was weighed immediately after removal to obtain the wet weight (WW). Subsequently, each part of the brain was baked at 100 ℃ for 48 h and record the dry weight (DW). The BWC was measured using the following formula: (WW-DW)/WW×100%.
Western blotting analysis
At each post-SAH time point, the brain samples were collected and prepared for western blot analysis. The left hemisphere sample was prepared using RIPA lysis buffer. 40 ng of protein sample was loaded onto an SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk and incubated with the following primary antibodies: rabbit anti-CCR4 (1:1000, GTX53474, Gene Tex); rabbit anti-CCL17 (1:1000, ab182793, Abcam); rabbit anti-Akt (1:1000, 9272S, Cell Signaling Technology); rabbit anti-phosphorylated AKT ser473 (1:1000, 4060S, Cell Signaling Technology); rabbit anti-PKCα (1:000, 2056S, Cell Signaling Technology); rabbit anti-phosphorylated PKCα (1:1000, ab180848, Abcam); rabbit anti- PKC (1:1000, (ab179522, Abcam); rabbit anti-phosphorylated 4EBP1 (1:1000, 2855S, Cell Signaling Technology), rabbit anti-4EBP1 (1:1000, 9644S, Cell Signaling Technology); rabbit anti-phosphorylated STAT6 (1:1000, 5397S, Cell Signaling Technology); rabbit anti- STAT6 (1:1000, 56554S, Cell Signaling Technology); and rabbit anti-β-actin (1:5000, 3700S, Cell Signaling Technology) overnight at 4 °C. Membranes were incubated with corresponding secondary antibody (1: 10000, Santa Cruz; 1:5000, Abcam) for 2 h at room temperature.
RNA isolation and quantitative PCR
Preparation of mRNA and real-time quantitive PCR was previously described 15. Total RNA was extracted from left hemisphere samples and cells using TRIzol reagent (Invitrogen) after washing with PBS. cDNA was synthesized from purified RNA using a SuperScript III First-Strand cDNA synthesis system (18080051, Life Technologies) according to the manuscript’s instructions. SYBR Green PCR Master Mix (Applied Biosystem, CA, USA) was used for PCR amplification and a real-time PCR machine (iQ5, Bio-Rad Laboratories) was used to quantify the expression of mRNAs. β-actin was used as an endogenous control and the expression levels were quantified using 2-△△Ct method. All prime sequences are listed in Supplementary Table 1, and detection of each primer was performed in the triplicate.
Immunofluorescence staining
Animals were anesthetized and perfused with 200 ml PBS followed by 50 mL10% formalin transcardially at 24 h after SAH. Brain samples were fixed in 10% formalin for 2 days. 30% sucrose was used to dehydrate the brain for a further 3 days. Brain sections were prepared as 10 μm slices. Samples were co-incubated with primary antibodies at 4 ℃ overnight anti-CCL17 (1:100, LS-C198166, LifeSpan BioSciences), anti-CCR4 (1:100, ab59550, Abcam), anti-CD206 (1:100, sc58986, Santa crzu biotechnology), anti-Iba-1 (1:100, ab178847, Abcam), anti-GFAP (1:200, ab53554, Abcam), and anti-NeuN (1:200, ab177487, Abcam). Slides were incubated with the appropriate secondary antibodies and observed using a fluorescence microscope (Leica Microsystems, USA).
TUNEL staining
According to the manufacturer’s instruction, staining of TUNEL was applied to quantify cell apoptosis with in situ Apoptosis Detection Kit (Roche, Indianapolis, USA) at 24 h post-SAH. The number of TUNEL-positive cells was analyzed in the hemorrhagic region. Four random visual fields per slide were observed by a blinded observer using a microscope at 200x magnification.
FJC staining
The number of degenerating neurons was assessed by FJC staining using a modified FJC Ready-to Dilute Staining Kit (Millipore, Billerica, MA, USA) at 24 h post-SAH. In accordance with the manufacturer’s instructions, slides were washed with PBS incubated with the FJC working solution for 20 min, and then visualized using a fluorescence microscope. The FJC-positive neurons in four parts of the hippocampus of each brain were manually counted using a microscope at 200x magnification and the ImageJ software (ImageJ 1.5, NIH, USA).
Stereotaxic cerebroventricular AAV-Rictor injection
Rats were anesthetized and then mounted onto a stereotaxic apparatus (Stoelting Instruments, Wood Dale, IL, USA). The AAV-CD68-shRictor (1.3×1012 TU/ml) was injected into both of the lateral cerebral ventricles (0.5 μl each; AP: -1.5 mm, ML:+/-1.1 mm, DV:-4.5 mm, relative to Bregma). The injection speed was 0.1 μl/min using an injection pump (Harvard Apparatus, Holliston,
MA, USA) equipped with a 1-μl Hamilton syringe. Before withdrawing the syringe, it remained in place for at least five minutes. SAH induction was performed 3 weeks after injection.
Statistical analysis
Statistical analysis were performed using SPSS analysis tools (IBM Corp., USA) or Prism9 software program (GraphPad Software, USA). All data are presented as the mean ± standard error (SEM). To calculate statistical significance between the two groups, a two-tailed unpaired Student’s t-test (for parametric analysis) or Mann-Whitney U test (for non-parametric analysis) was performed. One-way analysis of variance (ANOVA) followed by Tukey’s or Dunnett’s multiple comparisons test were used to detect differences in the results between groups. P values less than 0.05 were considered statistically significant. Individual in vitro experiments were performed at least two times with similar results. For in vivo experiments, data were collected from multiple independent experiments performed on different days with different rats.