Cells and cell culture
PC12 cells were purchased from the Kunming Institute of Zoology (Kunming, China). To transform PC12 cells into neuron-like cells, the cells were differentiated with 50 ng/mL nerve growth factor (NGF, 450-01, PeproTech, Rocky Hill, USA) for 2 days before the experiments. These cells were seeded into flasks (density of 1 × 104 cells/mL) in Dulbecco’s minimal Eagle medium (DMEM, 8119174, Gibco, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, 1989478, Gibco, Penrose, Auckland), 100 U/mL penicillin and 100 µg/mL streptomycin (Genom, GNM15140, Hangzhou, China). Cells were cultured at 37°C in a humidified incubator with 5% CO2.
SH-SY5Y cells were purchased from Procell (CL-0208, Wuhan, China). SH-SY5Y cells were maintained in Dulbecco’s modified Eagle medium: nutrient mixture F-12 (DMEM/F-12, Meilun Biotechnology, MAO214-Mar-19E (R), Dalian, China) in flasks (density of 1 × 104 cells/mL) supplemented with 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37°C in a humidified incubator with 5% CO2 for 7 days before the experiments [21, 22].
Cortical primary neurons were isolated from one-day-old Sprague–Dawley rats according to a previously published protocol with some modifications . Briefly, the newborn rats were sacrificed and their brains were collected. Cortices were isolated, removed and placed in a Petri dish. Meninges and blood vessels surrounding the brain tissue were removed under a microscope. The left hemisphere cortices were quickly moved to a 15 mL centrifuge tube filled with 5 mL Neurobasal Medium (2073081, Gibco, NY, USA) containing 2% B27 (17504044, Thermo Fisher Scientific, MA, USA) and 0.5 µM L-glutamine (Genom, GNM21051, Hangzhou, China). Samples were minced and homogenized and the solution was left to stand for 1 min. The supernatant was discarded, and 2 mL Neurobasal Medium with 0.5% FBS, 2% B27 and 0.5 µM L-glutamine was added to the centrifuge tube. The solution with brain tissue was mechanically dissociated into a single cell suspension by mixing with a plastic straw 20 times. After funnel filtration, the single cell suspension with the highest purity was cultured at 37°C in a humidified incubator with 5% CO2. Primary neurons were cultured for 8 days before experiments.
Oxygen And Glucose Deprivation (ogd) Model
The culture medium was changed to glucose-free DMEM (11966025, Gibco, NY, USA) without FBS and cells were placed in an anaerobic, temperature-controlled (37°C) chamber flushed with 5% CO2 and 95% N2 (v/v) for different durations according to the cell type and treatment (12 h for PC12 and SH-SY5Y cells, and 6 h for primary neurons).
Then, PC12 and SH-SY5Y cells were removed from the incubator, cultured with the normal DMEM (8119174, Gibco, NY, USA) containing 10% FBS (1989478, Gibco, Penrose, Auckland) and 1 g/L glucose, and returned to the incubator under normoxic conditions (37°C, 5% CO2) for 24 h, resulting in reperfusion. The primary neurons were cultured with Neurobasal Medium containing 0.5% FBS (1989478, Gibco, Penrose, Auckland), 2% B27 and 0.5 µM L-glutamine in an incubator under normoxic conditions (37°C, 5% CO2) for 24 h. The cells were collected for experiments after reperfusion.
Experimental groups and drug treatmentin vitro
Both IGF-1 (recombinant human IGF-1, Intergen, NY, USA) and LY294002 (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (DMSO). First, we wanted to explore the effects of different IGF-1 concentrations in three kinds of cells. The different concentrations administered in different cells were as follows: 5, 10, 50, 100, and 200 ng/mL  for both PC12 and SH-SY5Y cells that underwent OGD for 12 h, and 5, 15, 25, and 50 ng/mL  for primary neurons that underwent OGD for 6 h. Cell Counting Kit-8 (CCK-8) assay was used to assess cell viability of the three kinds of cells at different concentrations of IGF-1 to determine the optimal IGF-1 concentrations in PC12 cells, SH-SY5Y cells and primary neurons.
Then, we defined several groups to detect the effects of IGF-1 on cells that underwent OGD. In the Normoxia group, cells (1 × 106 cells/mL) were cultured under normal conditions for 24 h without OGD. In the OGD group, PC12 and SH-SY5Y cells (1 × 106 cells/mL) underwent OGD for 12 h and primary neurons (1 × 106 cells/mL) underwent OGD for 6 h. In the OGD + DMSO group, PC12 and SH-SY5Y cells were administered with DMSO and simultaneously underwent OGD for 12 h, and primary neurons were administered with DMSO and simultaneously underwent OGD for 6 h. In the OGD + IGF-1 group, PC12 and SH-SY5Y cells were administered with IGF-1 (100 ng/mL) and simultaneously underwent OGD for 12 h, and primary neurons were administered with IGF-1 (25 ng/mL) and simultaneously underwent OGD for 6 h. In the OGD + IGF-1 + LY294002 group, PC12 and SH-SY5Y cells were administered with both IGF-1 (100 ng/mL) and LY294002 (10 µM for PC12 cells  and 30 µM for SH-SY5Y cells  ) and simultaneously underwent OGD for 12 h, and primary neurons were administered with IGF-1(25 ng/mL) and LY294002 (10 µM)  and simultaneously underwent OGD for 6 h.
Cells were seeded at a suitable density in 96-well plates (10000 cells/well) and cell viability was measured using a CCK-8 assay (Meilun Biotechnology, Dalian, China) according to the manufacturer’s instructions. CCK-8 solution was added to the culture medium, and cells were incubated for 4 h at 37°C in a humidified incubator with 5% CO2. The absorbance was measured at 450 nm using a Microplate Reader (Bio-Rad, Hercules, CA, USA).
Adult male Sprague-Dawley rats, weighing 240–270 g, were purchased from the Wuhan University Center for Animal Experiments and housed under controlled conditions (3 rats per cage, 22–25°C, 50–60% relative humidity, and 12 h light/dark cycle) with free access to water and food. The rats were allowed to adjust to the environment for 3 days before the surgery, and their weights were properly controlled. The protocols used were approved by the Institutional Animal Care and Use Committee of Wuhan University. All procedures adhered to the regulations specified by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of rats used, as well as their suffering and mortality. The investigators were blinded to the treatment status of the animals during all surgical procedures.
Middle cerebral artery occlusion (MCAO) model
We established the MCAO model as described in our previous studies [23, 28]. The rats were anaesthetized with 4% isoflurane followed by an incision approximately 15 mm in the middle of the neck. A silicone-coated nylon monofilament (SunbioBiotech, Beijing, China) with a diameter of 0.45 mm was gently inserted via a bevel incision into the left external carotid artery (ECA) to a point approximately 18.5 ± 0.5 mm away from the bifurcation of the left carotid artery (CCA), where resistance was felt. The monofilament was fixed by suture and the connective tissue and skin were stitched up. After 90 min, the monofilament was removed for reperfusion injury and the external carotid artery was ligated. Rats that underwent the surgery were housed at a constant 25°C (temperature was maintained by an electric heater) with free access to water and food. The brains were harvested for experiments 24 h after surgery.
Experimental groups and drug treatmentin vivo
Both IGF-1 (recombinant human IGF-1, Intergen, NY, USA) and LY294002 (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in DMSO. In the Sham group, rats underwent 4% isoflurane anesthesia, identical surgery and lateral ventricle injection except for the insertion of monofilament, in that it was withdrawn immediately. In the MCAO group, rats underwent I/R injury. In the MCAO + DMSO group, rats were given DMSO (5 µL) 30 min before the MCAO surgery through a lateral ventricle injection (using the bregma as the anatomical reference point: anteroposterior, 0.8 mm; lateral, 1.5 mm; depth, 3.5 mm) at the rate of 1.0 µL/min by 10 µL microsyringe. In the MCAO + IGF-1 group, rats were given IGF-1 (0.1 mg, 5 µL)  30 min before the MCAO surgery through a lateral ventricle injection at the rate of 1.0 µL /min. In the MCAO + IGF-1 + LY294002 group, rats were given 5 µL DMSO solution, in which IGF-1 (0.1 mg) and LY294002 (50 mM)  were dissolved 30 min before the MCAO surgery, through a lateral ventricle injection at the rate of 1.0 µL/min.
Neurological deficit score
The neurological examination was performed 24 h after MCAO surgery using a previously published modified neurologic scoring system [31, 32] as follows: 0, no deficits; 1, difficulty in fully extending the contralateral forelimb; 2, inability to extend the contralateral forelimb; 3, mild circling toward the contralateral side; 4, severe circling; and 5, falling onto the contralateral side. A higher score indicates a more critical I/R injury.
Triphenyltetrazolium chloride (TTC) staining
The cerebral infarct volume was evaluated by 2% 2, 3, 5-triphenyltetrazolium chloride (TTC, Sigma, St. Louis, MO, USA; dissolved in phosphate buffer saline) [33, 34] staining. Rats were anesthetized by 5% isoflurane 24 h after the MCAO surgery, and brains were collected quickly. A 2 mm section of the frontal pole was discarded, and the remaining brain tissue was sliced into eight coronal sections approximately 2 mm in thickness using a sharp blade. Each slice was submerged into 2% TTC solution and stained at 37°C for 30 min in the dark. Finally, 4% paraformaldehyde was poured over to immerse the brain sections overnight in a fridge maintained at 4°C.
Normal tissue was stained bright red, while the infarct area remained pale gray. The stained tissues were imaged and analyzed using Image J software (National Institutes of Health, Bethesda, MD, USA). The infarct volume percentage was calculated as the infarct area of the ipsilateral hemisphere/total area of ipsilateral hemisphere × 100% .
TdT-mediated dUTP Nick-End Labeling (TUNEL) assay, immunofluorescence, Hematoxylin-eosin (H&E) staining and Nissl staining
As soon as rats had been sacrificed, the brains were removed and quickly frozen at -80℃. Brains were fixed by 4% paraformaldehyde for 24 h, and then dehydrated, embedded in transparent, dipped paraffin wax, and sliced (10 µm-thick coronal sections). Then, the TUNEL assay was applied according to the manufacturer’s instructions. Three stained sections were collected from the core ischemic area of every rat. Five different fields in each section were randomly selected for analysis under a microscope at 400 × magnification. The percentage of TUNEL-positive nuclei in the region was calculated to evaluate apoptosis.
Immunofluorescence was performed as described in our previous studies [19, 35]. The paraffin sections were incubated with the following primary antibodies: rabbit polyclonal anti- GFAP antibody (1:200, 17490-1-AP, Santa Cruz); rabbit polyclonal anti- IBA-1 antibody (1:200, 019-19741, Wako); mouse monoclonal anti- IGF-1 antibody (1:200, NBP2-34361, Novus Biologicals); and rabbit polyclonal anti-MAP2 antibody (1:200, 17490-1-AP, Santa Cruz,). Images were collected by fluorescence microscopy at 200 × or 400 × magnification.
For H&E and Nissl staining, the paraffin sections were stained with H&E and toluidine blue (Nissl staining) reagent at room temperature for 10 min. An observer who was blinded to the experiment counted the number of normal neurons and viable neurons in the three different fields. Pathological changes were observed using a light microscope at 40 × magnification.
Cells were lysed with RIPA lysis buffer on ice for 30min. Lysates were centrifuged at 4°C and 12000 × g for 15 min and supernatants were collected. Brain tissue was cut into pieces with small scissors and washed several times with 0.9% saline at 4°C. The brain tissue was grinded into pulp with a grinding rod, and the pulp was transferred to a 10 mL centrifugal tube. Tissue RIPA lysis buffer and Phenylmethanesulfonyl Fluoride (PMSF) were added and the solution was lysed on ice for 30 min. The solution was blown repeatedly with a micro pipette to ensure that samples were completely lysed. Lysates were centrifuged at 4°C and 14000 × g for 20 min and the supernatants were collected.
To each protein sample, 5 × sample buffer was added at a ratio of 4:1. Samples were heated at 100°C for 10min and β-mercaptoethanol was added.
Western blot was performed as described in our previous work . Briefly, after SDS-PAGE, the proteins were transferred to a PVDF membrane (Millipore, Billerica, MA, USA). Western blot analysis was performed with the following antibodies: rabbit polyclonal anti-GAPDH antibody (1:2000, 10494-1-AP, Santa Cruz Biotechnology, Dallas, TX, USA); rabbit polyclonal anti-β-actin antibody (1:2000, ab8227, Abcam, Cambridge, MA, USA); rabbit polyclonal anti-PI3K antibody (1:2000, ab191606, Abcam, Cambridge, MA, USA); rabbit polyclonal anti-p-AKT antibody (1:2000 ab8805, Abcam, Cambridge, MA, USA); rabbit polyclonal anti-YAP antibody (1:1000, ab76252, Abcam, Cambridge, MA, USA); rabbit monoclonal anti-TAZ antibody (1:1000, ab84927, Abcam, Cambridge, MA, USA); and rabbit polyclonal anti-Caspase 3 antibody (1:1000, ab13847, Abcam, Cambridge, MA, USA).
The membrane was then incubated with a secondary goat anti-rabbit antibody (1:1000, ab6721, Abcam, Cambridge, MA, USA) for 2 h at room temperature. The protein electrophoresis bands were imaged using an Odyssey infrared scanner (LICOR Bioscience, Lincoln, NE, USA) and analyzed using Image J software.
The data were analyzed with the SPSS for Windows 20.0 software package and GraphPad Prism Version 6.0 software (GraphPad Software Inc., La Jolla, CA, USA) and were expressed as means ± SD. The images were analyzed by Image J software. Differences among groups were determined by performing a one-way ANOVA, followed by Turkey posthoc tests. Two-tailed t tests were used for comparisons between two groups. A two-tailed p value < 0.05 was considered statistically significant.