Animals
The experimental designs of this study were approved by the institutional Animal Care and Use Committee of the First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province. We performed all animal procedures strictly according to the National Institutes of Health guidelines. The results of animal experiments were reported according to AARIVE (Animal Research: Reporting in vivo Experiments). Male Sprague-Dawley (SD) rats (weight: 250–300 g) were purchased from the Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China). All rats were housed in the applicable environment under regular light alternates with dark (12h/12h) cycle and unlimited access to water and food. We minimized the numbers of rats that were used for the experiments as much as possible.
CSF collection
The experimental designs of this study were approved by the Medical Ethics Committee of the First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province. 500µl CSF were collected from 10 patients with normal pressure hydrocephalus who need routine lumbar puncture drainage test after admission. Moreover, 500µl CSF from patients with ICH were obtained during the operation. During the removal of the hematoma via the lateral fissure approach, the outflow of CSF was quickly absorbed by a syringe. The specific information of patients is shown in Table S1. [19].
Establishment of the experimental ICH model in rats
The experimental ICH model was conducted in vivo by injection of autologous-arterial blood [3]. Briefly, rats were anaesthetized by inhalation of 5% isoflurane initially. After successful anesthetization, SD rats were fixed in a prone position on the stereotaxic frame (Zhenghua Biological Equipment Co. Ltd., Anhui, China). The periosteum was exposed after the median scalp was incised under aseptic conditions; we drilled a hole above the right basal ganglia (3.5 mm to the right and 0.2 mm posterior to the bregma). A total of 150 μl autologous rat blood was obtained from the caudal artery, then a microsyringe equipped with 100 μl autologous-arterial blood was slowly inserted to a depth of 5.5 mm and injected (20 μl/min) into the right basal ganglia. The position of the needle was maintained after the injection was completed. Bone wax was used to burr the hole and the skin incision was sutured. The heart rate of the animal was monitored and the body temperature was maintained at 37 ± 1°C during the surgical procedure. The rats in the sham group underwent the same procedure, with the exception that an equal volume of saline solution was injected instead of blood.
Experimental design
This experiment was divided into six parts (Figure S1). In experiment 1, we collected CSF from 10 patients with normal pressure hydrocephalus and 16 patients with ICH. All of the samples were detected by ELISA. Experiment 2 was performed to further investigate the changes in the protein level of SEMA4C and Plexin B2 after ICH. Additionally, the relationship between SEMA4C and Plexin B2 after ICH were verified by co-immunoprecipitation (Co-IP) testing. To evaluate the effect of recombinant SEMA4C after ICH, Rats were randomly divided into five groups in experiment 3: sham, ICH + vehicle (phosphate buffered saline [PBS]), ICH + rSEMA4C (0.15 μg/kg), ICH + rSEMA4C (0.5 μg/kg), and ICH + rSEMA4C (1.5 μg/kg). rSEMA4C was administered by intraventricular injection at 1 h post-ICH. Based on the outcomes, a dose of 0.5 μg/kg was found to be optimal for the following experiments.
Based on the experiments above, the following experiments exploited the time period at 24 h after ICH. In experiment 4 and 5, rats were randomly average divided into seven groups: sham, ICH, ICH + Vehicle, ICH + rSEMA4C, ICH + LV-Con, ICH + LV-SEMA4C, and ICH + LV-SEMA4C-secr. At 24 h after ICH,,rats were uesed for terminal deoxynucleotidy1 transferase-mediated dUTP nick-end labeling (TUNEL) staining, fluoro- Jade B (FJB) staining, and ethological testing. In experiment 6, primary-cultured neurons were used; enriched neurons were exposed to oxyhemoglobin (OxyHb) to simulate ICH in vitro. Neurons were divided into five groups as follows: Control, OxyHb, OxyHb + LV-Con, OxyHb + LV-SEMA4C, and OxyHb + LV-SEMA4C-secr. After 24 h of OxyHb exposure, the cells were collected for immunofluorescent staining, GLISA, and live-death cell-viability assay. The sum mary of experiment groups and mortality rate are showed in Table S2.
ELISA
The human CSF samples were collected to detect the levels of sSEMA4C using specific ELISA kits (Fusheng Industril, Shanghai, China) according to the manufacturers’ instructions.
Primary cortical neurons cultures
Cortical neurons were isolated from primary rat embryos and cultured as described previously [20]. Briefly, cortical tissues of 18th-day rat embryos were isolated from the whole brain after removing the meninges and blood vessels. Next, 0.25% trypsin-EDTA solution was used to digest the cortical tissues for 5–8 min at 37°C. Then, the supernatant was centrifuged at 1500 r/min for 5 min, followed by filtration of the brain suspension. Dissociated neurons were plated onto 6- or 12-well plates (Corning, USA) precoated with 0.1 mg/ml poly-D-lysine (Sigma, USA), cultured in Neurobasal medium supplemented with 2% B-27 and 0.5 mM GlutaMAX TM- I (all Gibco, Carlsbad, CA, USA), and maintained at 37°C under humidified conditions and 5% CO2. Half of the medium was changed every two days. Based on previous experience [21], Oxygen hemoglobin (OxyHb) was used to stimulate ICH injury in vitro, primary cortical neurons were incubated with OxyHb (20 μM) at 37 °C and 5% CO2 for 24 h.
Antibodies and drugs
Anti-SEMA4C antibody (sc-136445) and Anti-ID-3 antibody (sc-56712) were from Santa Cruz Biotechnology. Rb pAb to NeuN (ab104225), Anti-ID-1 antibody (ab230679), and Recombinant human semaphorin 4c (ab162889) were purchased from Abcam. Anti-Plexin B2 antibody (Fnab06545) was from FineTest. Cleaved-caspase-3 antibody (BF0711), β-tubulin antibody (AF7011), and GAPDH antibody (AF7021) were from Affinity Biosciences. Protein A/G immunoprecipitated magnetic beads (B23202) were from Bimake. Normal rabbit IgG (2729) was from Cell Signaling Technology. RhoA G-LISA Activation Assay Kit (Colorimetric Format) (BK124) was from Cytoskeleton, Inc. Human semaphorin 4c ELISA kits (A100745-96) were from Shanghai Fusheng Industrial Co., Ltd. GM6001 (A13320) was purchased from AdooQ BioScience. Secondary antibodies for western blot analysis, including goat anti-mouse IgGHRP (sc-2005) and goat anti-rabbit IgG-HRP (sc-2004), were obtained from Santa Cruz Biotechnology. Secondary antibodies for immunofluorescence analysis, including Alexa Fluor-488 donkey anti-rabbit IgG antibody (A21206), Alexa Fluor-555 donkey anti-rabbit IgG antibody (A31572), and Alexa Fluor-555 donkey anti-mouse IgG antibody (A31570), were purchased from Invitrogen.
Lentiviral transduction
Full-length rat SEMA4C cDNA was obtained from www.uniprot.org:D4A9J3, and its mutant, containing secretory extracellular domain (SEMA4C-secr), was generated by GeneChem Company (China). The sequence elements of lentiviral vector were Ubi-MCS-3FLAG-SV40-puromycin (Table S3). The titer of LV-SEMA4C was 2.8 × 108 TU/ml and the titer of LV-SEMA4C-secr was 1 × 108 TU/ml. In vivo, intraventricular injection of lentivirus was conducted according to the methods outlined in a previous study [22]. Briefly, 10 days before ICH surgery, SD rats were fixed on a stereotaxic apparatus after anesthesia. The amount of lentivirus injected in each rat was 5 × 105 TU. A left side hole was drilled 1.5 mm lateral and 1.1 mm posterior to the bregma, and lentivirus was slowly infused into the lateral ventricle (3.5-mm depth) at a speed of 0.5 μl/min. The microsyringe was withdrawn slowly 5 min after injection to avoid reflux. The burr hole was closed with bone wax. In vitro, primary neurons were transduced with moderate LV (multiplicity of infection [MOI] = 10), and addition of 20 μl HA (HitransG A, GeneChem Co., Shanghai) enhanced the transduction solution 5–7 days after successful extraction.
Drug administration
According to a previous study [23], GM6001 (a broad-spectrum MMP inhibitor) was dissolved in DMSO and diluted to the final concentration with 0.9% normal saline. GM6001 (50 mg/kg) or equal volume of vehicle was administered intraperitoneally 2 h after the induction of ICH. In vivo, recombinant human semaphorin 4C was injected into the left lateral ventricles 1 h after ICH induction. The intracerebroventricular injection was performed as described above. Three doses of recombinant human semaphorin 4C were tested (0.15 μg/kg, 0.5 μg/kg, and 1.5 μg/kg).
Western blot analysis
The brains of rats were isolated after lavage with PBS, and the cerebral cortex surrounding the hematoma was collected. Cortical tissue was mechanically lysed in a lysis buffer. The supernatant was discarded after the lysed cortical tissue was centrifuged (12,000 r/min, 4°C, 5min). The final protein concentration was measured using the BCA protein Assay Kit (Beyotime, China). Molecular weight makers (Thermo Fisher Scientific, Waltham, MA, USA) and an equal volume of protein samples were loaded on sodium dodecyl sulfate (SDS)-polyacrylamide gel, separated, and electro-transferred onto a nitrocellulose membrane (Millipore Corporation, Billerica, MA, USA). Then, the membrane was blocked in 5% bovine serum albumin (BSA) blocking buffer for 1 h at room temperature. Subsequently, the membrane was incubated overnight at 4°C with primary antibodies against SEMA4C, Plexin B2, ID1, ID3, and cleaved caspase-3. Subsequently, the membrane was incubated with the corresponding HPR-conjugated secondary antibodies for 1 h at room temperature. After washing three times with PBST, the protein bands were detected by an enhanced chemiluminescence (ECL) kit (Beyotime, China). ImageJ software (NIH, Bethesda, MA, USA) was used to analyze the density of the protein bands. β-tubulin or GAPDH functioned as a loading control.
Co-immunoprecipitation (Co-IP)
The sample of brain tissue was prepared as described for western blotting. Briefly, brain samples were ground in a cell lysis buffer (Beyotime, China) and centrifuged to obtain the supernatant. Each supernatant was rotated for 1 h at 4°C after addition of Protein A/G immunoprecipitated magnetic beads. The samples were centrifuged again, and the supernatant was collected. Anti-Plexin B2 antibody or normal IgG were combined into the supernatant and rotated overnight at 4°C. Thereafter, the beads were added and rotated for a further 4 h at 4°C. The protein-antibody-bead mixture was centrifuged and the supernatant was removed. Then, the precipitate was denatured with the SDS loading buffer. Finally, the protein was further detected by western blotting.
TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining
TUNEL staining was utilized to quantity cellular apoptosis around the hematoma according to the manufacturer’s protocol. The TUNEL-positive cells in the area of interest were observed and analyzed using a fluorescence microscope (OLYMPUS BX50/BX-FLA/DP70; Olympus Co., Japan). An observer who was blinded to the group allocation was responsible for counting and analyzing the positive cells.
Fluoro-Jade B (FJB) staining
FJB is a classic stain that is used to detect degenerated cells in brain tissue. The brain sections were dewaxed and rehydrated, and then transferred to a 0.06% KMnO4 solution in the dark for 15 min at room temperature. After washing three times, the slides were immersed in 0.0004% FJB working solution for 20 min. Subsequently, the slides were incubated in an incubator (50–60°C) for 30min, and enveloped with neutral resins. Finally, the sections were surveyed by a fluorescence microscope.
Immunofluorescence analysis
In the in vivo experiments, the brain tissue was fixed, embedded, and cut into 4-μm sections. In the in vitro experiments, cultured neurons were fixed. The specific methods have been described previously [24]. Laconically, sections and cells were incubated with primary antibodies and then homologous secondary antibodies. The nuclei were stained with DAPI mounting medium. Lastly, a fluorescence microscope was used for observing the sections and cells. Quantitative analysis was performed by observers who were blinded to the group allocation.
Cell-viability assay
A cell-viability assay (Invitrogen, USA) was performed according to the manufacturer’s instructions. Briefly, green dots (membrane-permeant calcein AM) indicated live cells and red dots (fluorescent ethidium homodimer-1) represented dead cells. The cells in three random fields were counted by a blinded observer. The experiments were repeated three times.
RhoA activity assay
GTP-bound RhoA was measured using G-LISA Activation Assay Kits (Cytoskeleton), according to the method reported previously [25, 26]. Following stimulation, the cells were washed three times with cold PBS and lysed with lysis buffer for 15 min on ice. The mixture was centrifuged at 10000 × g for 1 min at 4°C. Then, the supernatants were aliquoted, snap-frozen in liquid nitrogen, and stored at −80°C. GTP-bound RhoA activity was evaluated following the manufacturer’s instructions.
Brain water content
Rats were administered intraperitoneally with 4% chloral hydrate 24 h after ICH. The total brain tissue was removed immediately, and the brain was separated into two hemispheres symmetrically; each hemisphere was further divided into two parts containing the cortex and basal ganglia. The final samples consisted of five parts: the ipsilateral basal ganglia (Ipsi-BG), the ipsilateral cortex (Ipsi-CX), the contralateral basal ganglia (Cont-BG), the contralateral cortex (Cont-CX), and the cerebellum (CB). The wet samples were weighed and recorded immediately. Then, the samples were moved to a thermostatic drier at 100°C for 72 h. The dried samples were also weighed and recorded, and the value of (wet weight – dry weight)/ (wet weight) × 100% was calculated.
Modified Garcia score test
The modified Garcia score was recorded as previously described [27]. The score consists of seven individual tests that assess spontaneous activity, axial sensation, vibrissae proprioception, symmetry of limb movement, lateral turning, forelimb walking, and climbing. Each subtest was given a score ranging from 0 to 3, with a maximum neurological score of 21.
Adhesive-removal test
The adhesive-removal test was used to access motor coordination and sensory function of the rat forelimbs after ICH [28]. The rats were placed in a cage for a few min to become familiar with the testing environment, and then a 9-mm circular sticker was attached to the palms of the bilateral forelimbs of the rats. The rats received training, which involved removing both stickers from the forepaws, for three days before ICH. After the surgery, the test was performed on days 1, 7, 14, and 20, and the time was recorded by two blinded observers.
Rotarod test
The rotarod test was used to evaluate the motor function of rats [29, 30]. The rats were initially placed onto the (10-cm diameter) (ZH-300B, Anhui Zhenghua Biological Equipment Co. Ltd., Anhui, China). The initial speed was 5 rpm, and was increased by 0.5 rpm per second. The rotarod cylinder stopped automatically at 1 min. Each rat received for three days, three times a day. The time at which the rats dropped or gripped the rotarod spindle and spun for two consecutive revolutions was recorded. After ICH surgery, the test was performed on days 1, 7, 14, and 20. Two blinded researchers were responsible for data recording.
Morris water maze
Morris water maze tests (swim distance and escape latency) were performed to access spatial cognitive functional deficits on days 21 to 25 after ICH. The Morris water maze test probe quadrant duration was measured on day 25 after ICH [31]. A round pool (180-cm diameter) filled with opaque water fell into four quadrants. A platform (10-cm diameter) was submerged in the pool at a depth of 1.5 cm and was set at a fixed position (the fourth quadrant). During the initial training phase, the rats were placed in the pool and given 60 s to find the fixed platform. Rats that were unable to find the platform in the stipulated time were induced to the platform and allowed to stay for 10 s to intensify their learning and memory. ICH surgery was performed after five days of training. Escape latency (EL) referred to the time that the rats took to reach the target platform from the starting point. During the formal experiments, the rats were placed in the second quadrant and allowed to find the platform. Both ELs and swimming distances were recorded on days 21 to 25; then, on the 26th day after the above experiments, the platform was removed. The rats were placed in the second quadrant again, and the number of times that the rats swam through the previous platform within 60 s was recorded to evaluate the spatial memory. The swimming speed of each test was recorded to assess the general locomotor functions of rats. The room temperature was maintained at 25 ± 2°C, and the water temperature was maintained at 20 ± 2°C during the entire process.
Statistical analysis
All data were analyzed by GraphPad Prism 8.0 software (GraphPad, San Diego, CA, USA) and presented as the mean ± SD. All data were tested for normality, and two-sided unpaired Student’s t-test was used to compare the two groups. The correlation between two variables was determined by Pearson’s correlation test. A one- or two-way ANOVA was adopted to compare differences between groups. Tukey’s multiple comparisons have been conducted for multiple testing. P-values < 0.05 were considered to indicate a statistically significant difference. Detailed statistics were shown in Table S4.