Male C57BL/6 mice, 8–10 weeks old (25~30 g), were purchased from Shanghai JieSiJie Laboratory Animal Co., Ltd. Mice were housed in a 12-h light/dark cycle at a constant temperature and humidity controlled room for a minimum of 3 days before surgery, with free access to food and water. All animal experiments in the study were approved by the Animal Care and Use Committee of Shanghai Medical College, Fudan University.
In the present study, all mice were randomly assigned to the following experiments. The experimental design was shown in Part 1 of the Supplementary material-1 (Figure S1). The summary of experimental groups, animal numbers and mortality rate in the study was listed in Part 2 of the Supplementary material-1 (Table S1).
ICH surgery was induced by stereotactic-guided injection of autologous whole blood into the right basal ganglia as previously described . Briefly, mice were anesthetized with an isoflurane-oxygen mixture during the surgical procedure, and the body temperature was maintained at 37.0 ± 0.5 °C using a heating blanket. Mice were positioned prone on the stereotactic head frame (Kopf Instruments, Tujunga, CA, USA). An artificial tears ointment was applied to keep the eyes moist during surgery. Arterial blood was collected in a nonheparinized capillary tube and transferred immediately into a 27-gauge needle on a 250 μL Hamiton syringe. Acoronal incision was performed to expose the cranium until the bregma was clearly visible. After that, a 1-mm cranial burr hole was drilled in the skull, and the Hamilton syringe was inserted into the right basal ganglia in accordance with the stereotactic guide (coordinates: 0.2 mm posterior, 2.2 mm lateral to the bregma). Following this, a total volume of 30 μL autologousblood (initial 5 μL at depth of 3.0 mm below dura and followed by 25 μL at the depth of 3.5 mm below dura 5 minutes later) was infused using a microinfusion pump (Stoelting, Harvard Apparatus, Holliston, MA) at a rate of 2 μL/min. To prevent possible leakage due to blood backflow, the needle was left in place for an additional 10 min after the completion of 30 μL injection and slowly withdrawn at a rate of 1 mm/min. The burr hole was then sealed with sterilized medical bone wax and the incision of scalp was sutured. Mice were then allowed to recover fully on a heating pad at 37 ℃, and the neurological deficits were closely observed. The sham surgery was performed following the same procedure without blood injection.
Recombinant irisin (#100-65, Peprotech, USA) was dissolved in phosphate-buffered saline (PBS). Three differentdoses of irisin were tested (80 μg/kg, 250 μg/kg,and 750 μg/kg) and were administered intranasally at 30 minutes afterICH induction. Cilengitide trifluoroacetate (10 mg/kg, Selleck, USA), a selective inhibitor of αVβ3 and αVβ5 integrins, was dissolved in DMSO and administered intraperitoneally at 2 h before ICH induction. Dorsomorphin (5 μg/mouse,Sigma, MO), aselective AMPK inhibitor, was dissolved in DMSO and administered intracerebroventricularly (i.c.v.) 30 minutes before ICH injury .
Intracerebroventricular administration was performed as previously described . Briefly, a 1-mm cranial burr hole was drilled at the following coordinates relative to bregma: 0.22 mm posterior, 1.0 mm lateral). A 26-gauge needle of a 10 μLHamilton syringe was inserted into the left lateral ventricle through the cranial burr hole at the depth of 2.25 mm deep under dura. A microinfusion pump was used for intracerebroventricular injection at a rate of 0.667 μL/min. The needle was left in place for an additional 5 minutes at the end of infusion and then removed slowly over a 3-min period. The burr hole was immediately sealed with sterilized medical bone wax.
Short-term neurobehavioral assessment
Short-term neurofunctional behavior were assessed with modified Garcia score test, forelimb placement test and corner turn test at 24 h and 72 h post-ICH by an independent researcher who was blinded to the information of experimental groups, as previously described . The modified Garcia score was assessed by a 21-point score system with seven individual tests including spontaneous activity, axial sensation, vibrissae proprioception, limb symmetry, lateral turning, forelimb walking, and climbing. Each subtest was scored from 0 to 3, and the total score was generated through adding up the sum of seven subtest scores. For the forelimb placement test, the placement of left forelimb on the countertop when the vibrissa was stimulated was recorded. The percentage of the left forelimb placement out of ten consecutivevibrissae stimulations was calculated. The corner turn test was performed utilizing the device which consisted with two boards forming a 30° angle vertically on the platform. The mice were allowed to advance into a 30° angel corner and exit by turning either to the left or the right. Choice of turning was recorded for a total of ten trials, and the result was the percent of left turns in 10 trials.
Long-term neurobehavioral assessment
Rotarod test was performed to evaluate sensorimotor function, coordination, and balance on days 7, 14 and 21 post-ICH with 47650 Mouse Rota-Rod (UGO BASILE). The mice to be tested were placed in each lane on the rotating cylinder at a speed of5r/min and accelerated to 40r/min within 300s and went on rotating at the constant speed for 200 s (total 500 s).The falling latency which is defined as the time duration when a mouse stabilizes himself on the rotating cylinder without falling was recorded.Three trials were performed and the mean time of falling latency was recorded.Morris water maze was carried out to evaluate spatial learning and memory abilities on days 21 to 26 after ICH, as previously reported .In the learning phase of the test, the mouse was placed into the pool from one of the three quadrants without the platform and allowed to swim for up to 60 s to escape to the hidden platform. The time until the animal found the platform (escape latency) was recorded for each trial as “spatial learning”.At the end of each trial, the mouse was allowed to remain on the platform or placed on the platform (if the mouse could not find the platform within 60 s) for 10 s with prominent spatial cues displayed around the room. Mice were pre-trained for 3 consecutive days before ICH induction (3 trials on each day). After injury, 3 trials were conducted on each testing day for 5 consecutive days (21-25 days after ICH). In the memory phase of the test at 26 days after ICH, a single, 60-s probe trial was performed with the platform removed. The time the mouse spent swimming in the goal quadrant where the platform was previously located was recorded as “spatial memory” and expressed as a percentage of the total testing time of 60 s.
Brain water content measurement
Brain water content (BWC) was measured through wet/dry method as previously reported . Briefly, mice were euthanized by decapitation under deep anesthesia at 24 and 72 h post-ICH. Brians were removed immediately and divided into five parts: ipsilateral and contralateral cortex, ipsilateral and contralateral basal ganglia, and cerebellum.Each brain section was measured immediately on an analytical microbalance to obtain the wet weight (WW) and then dried for 24 h at 100 °C to obtain the dry weight (DW).Brain water content was calculated through the following formula: brain water content (%) = [(WW − DW)/WW] × 100%.
Enzyme-linked immunosorbent assay (ELISA)
Plasma levels of irisin were measured at 6 h and 24 h after injury following the manufacturer's instructions of a commercial ELISA kit(Phoenix Pharmaceutical, Burlingame, CA)with a 1:2dilution of each plasma sample (50 μL).Intra- and inter-assay variances were <4–6% and <8–10%, respectively, and the range of detectable concentrations was 0.066–1024 ng/ml.
Quantitative real-time polymerase chain reaction (q-PCR)
Total RNA was extracted from brain tissue 24 hours after ICHaround the hemorrhageusing Trizol (Qiagen, Hilden, Germany) according to the manufacturer’s protocol,after which RNA was reverse transcribed into cDNA using Superscript III First-Strand Synthesis SuperMix (Invitrogen, Carlsbad, CA, USA).Quantitative real-time polymerase chain reaction (q-PCR) was performed using synthetic primerslisted in Part 3 of the Supplementary material-1 and SYBR GREEN FAST mastermix (Qiagen).Data collection was performed on the RT-PCR System (Bio-Rad, Hercules, CA, USA). GAPDH was used as an internal control. The relative quantitation value for each gene was performed using the comparative cycle threshold method .
Western blot analysis
Mice were transcardially perfused with coldPBS under deep anesthesia at 24 h post-ICH. Brain samples were stored at -80 freezers after quickly extracted and snap frozen in liquid nitrogen.Western blotting was performed as previously described. Briefly, after brain samples homogenized usingRIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz,CA, USA) and centrifugedat 4 °C for 30 min at 14,000 rpm, equal amounts of protein were loaded on anSDS-PAGE gel and run using electrophoresis and thentransferred to a nitrocellulose membrane.Equal amounts of protein wereloaded on an SDS-PAGE gel and run using electrophoresis,then transferred to a nitrocellulose membrane.The membranewas blocked and then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-irisin (1:1000, ab174833, Abcam, USA); rabbit integrin αV (1:1000, ab179475, Abcam, USA); rabbit anti-integrin β5 (1:1000, #3629, Cell Signaling Technology, Inc., MA, USA); anti-AMPKα (1:1000, #5831, Cell Signaling Technology, Inc., MA, USA); anti-p-AMPKα (1:1000, #2535, Cell Signaling Technology, Inc., MA, USA); rabbit anti-IL-1β (1:1000, #12242, Cell Signaling Technology, Inc., MA, USA); rabbit anti-Iba-1 (1:1000, ab178846, Abcam, USA); rabbit anti-MPO (1:1000, ab208670, Abcam, USA); rabbit anti-TNF-α (1:1000, #11948, Cell Signaling Technology, Inc., MA, USA); rabbit anti-Bcl-2 (1:2000, ab182858, Abcam, USA), rabbit anti-Bax (1:4000, ab182733, Abcam, USA).The membranes were incubated with mouse anti-β-actin (1:2000,sc-47778, Santa Cruz, USA) as a loading control.Appropriate secondary antibodies (1:3000, Santa Cruz; 1:5000, Abcam) were selected to incubate with the membrane for 2 h at room temperature.The membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Proteintech) secondary antibodies.The bands wereprobed with an ECL Plus chemiluminescence regent Kit(Amersham Biosciences, Arlington Heights, PA, USA)and visualized with the image system (Versa Doc, model 4000, Bio-Rad, Hercules, CA, USA). Relative density ofthe protein immunoblot images were analyzed by ImageJsoftware (Image J 1.5, NIH, USA).
After anesthetized deeply at 24 h and 72 h after ICH, mice were transcardially perfused with icecold PBS and 4% paraformaldehyde. Brains were then removed and immersed in 4% paraformaldehyde, 20% sucrose, and 30% sucrose successively to complete fixation and dehydration. Coronal sections (25-μm thick) were sliced using the freezing microtome (HM525NX, ThermoFisher), and stored in tissue stock solution.After washed in PBS and PBS+0.3% Triton, coronal sections were incubated in PBS+1% Triton to break the cell membrane and blocked with 10% goat/donkey serum for 1 h. Coronal sections were incubated with primary antibodies overnight at 4 ℃.After being washed in PBS three times with 10 min intervals, the sections were incubated with secondary antibodies conjugated with Alexa Fluor-488/594/647 for 2 h at room temperature. Nuclear staining was performed with 4',6-diamidino-2-phenylindole (DAPI). Sections were then observed and imaged under a Nikon microscope (Nikon).
For quantification of neuronal apoptosis at 24 h after ICH, double staining of neuronmarker NeuN (red) and TUNEL (green) was conducted using in situ Apoptosis Detection Kit (Roche, Indianapolis, IN, USA) according to the manufacturer’s instructions .In the peri-hematoma area,TUNEL-positive neurons were counted manually andthe numbers of randomsix sections per brain slice over a microscopic field of 200 Í magnificationsusingImage J software (Image J 1.5, NIH, USA)were averaged. Data was expressedas the ratio of TUNEL-positive neurons (%).
Degenerating neurons wasevaluated by FJC staining using a modified FJC Ready-to-Dilute Staining Kit (Millipore, Billerica, MA, USA) at 24 h post-ICH as previously reported .According to manufacturer’s instructions,slides were washed in PBS incubated with the FJC working solution for 20 min, and then visualized using a fluorescence microscope (Leica Microsystems) in blinded manner.FJC-positive neurons were manually counted in the peri-hematoma regions of sixparts per brain at Í 200 magnification using ImageJ software (Image J 1.5, NIH, USA). The data were averaged and expressed as positive cells/mm2.
All data are expressed as mean ± standard deviation (SD).Data were normally distributed as tested using the D'Agostino and Pearson omnibus normality test (P > 0.05).For comparisons between 2 groups, the Student’s t-test was used for comparisons of variables with normal distribution from independent samples. Multiple comparisons were statistically analyzedusing One-way analysis of variance (ANOVA)followed by Tukey post hoc multiple comparison analysis. Two-way ANOVAfollowed by Tukey post hoc test, was used to compare the changes according to the different levels of multiple categorical variables (brain water content, long-term neurological function).Data analyses were conducted using Prism 9 (GraphPad Software, CA, USA). All statistical tests were two-sided, and a P<0.05 was considered statistically significant.