OPC preparation and cyclosporine injection
NSCs were generated from the CNS tissue of spontaneously aborted human female fetuses at gestational week 10-12 from the Pediatrics Laboratory of the Sixth Medical Center of PLA General Hospital (Beijing, China). Informed written consent was obtained from the mothers, and differentiated in vitro according to a modified version of a previously published protocol . Briefly, the brain was homogenized into a suspension of single cells by mechanical dissociation. Cells were trypsinized and transferred to a neural differentiation medium containing DMEM/F12, leukemia inhibitory factor, basic fibroblast growth factor, epidermal growth factor, and nonessential amino acids (all from Gibco, Waltham). Ten-day old neurospheres were used for this experiment. NSC isolation, culturing, and differentiation were performed as previously described. Purity and viability of transplanted NSCs was confirmed by cell morphology and immunofluorescence staining of specific markers nestin (mouse IgG1, 1:1000, Abcam), A2B5 (rabbit IgG, 1:200, Abcam), platelet-derived growth factor receptor (PDGFRα, rabbit IgG, 1:800, Cell Signaling Technology), neuron-glial antigen 2 (NG2, rabbit IgG, 1:200, Millipore), O4 (mouse IgG, 1:200, Sigma), and glial fibrillary acidic protein (GFAP, mouse IgG, 1:500, Santa Cruz). All transplant recipients received cyclosporine (i.p, 10 mg/kg) daily three days before transplantation and continuing for a total of four weeks. Subsequently, the rats were administered cyclosporine (100 μg/mL) via their drinking water until perfusion. During the immunosuppression period, the cyclosporine dose was appropriately reduced or discontinued if the rats were found to have sustained weight loss, red eyelids, or nasal damage.
Neonatal rat WMI model and treatment
Clean-grade SD rat pups (P3) obtained from the Center of Experimental Animals of Xuzhou Medical University were randomly divided into control (Ctrl), WMI and vehicle-treated (Vehicle) group, WMI with ZJU-37-treated (ZJU-37) group, WMI with OPC transplantation (OPCs) group and WMI with ZJU-37-treated and OPC transplantation (ZJU-37+OPCs) group (n=12 per group). Animals were housed in an air-conditioned room with a 12 h light/dark cycle, and provided with adequate food and water. All efforts to reduce the number of animals used and minimize animal suffering were made. All WMI groups were generated as described previously . Briefly, P3 pups were placed in a refrigerator at -20°C for 7-10 min. After freezing anesthesia, the right common carotid artery was carefully isolated from the surrounding tissue and ligated. Then, the wound was sutured with an 8-0 suture and the time of operation was controlled to within 5 min. Upon completion of the surgery, pups were moved to the recovery area under a heat lamp for 10 min, returned to their mother, and allowed to recover for an additional 1 h before exposure to 6% oxygen (94% nitrogen saturation) at 37°C for 90 min in a humidified chamber. After monitoring recovery, the pups were returned to cages to continue feeding. Sham-operated rats without hypoxia were used as the Ctrl group. At P12, the ZJU-37 group was injected intraperitoneally with ZJU-37 (i.p, 10 mg/kg) once daily for the first month, every other day for the second month and every third day for the third month until euthanasia. The Vehicle group was administered DMSO (i.p, 10 mg/kg) whereas rats in the Ctrl group received no treatment. At P4, P6, P8 and P10, the degree of injury (mild/moderate to severe) was determined by observing the general condition of animals, neurobehavioral evaluation, and histopathology.
Moderately to severely injury rats were fixed on a stereotaxic apparatus after narcotization with 4% chloral hydrate (4 mL/kg) at P12. A small incision was made through the midline to expose the skull. The anterior fontanelle was used as a guide to determine the puncture point whose coordinates from bregma were as follows: anteroposterior-1.0 mm; mediolateral-1.5 mm; dorsoventral-2.0 mm by brain stereotaxic apparatus. A 5 μL microsyringe was used to withdraw 3 μL cell suspension (approximately 3×105 OPCs), which was then slowly injected into the transplant site. After injection, the needle was left at the injected site for an additional 5 min then slowly withdrawn. Subsequently, the scalp wound was closed and the rats were placed back into home cages and nursed after fully awaking from anesthesia under a heat lamp. The transplantation time for each rat was approximately 30 min. In the ZJU-37+OPCs group, ZJU-37(i.p, 10 mg/mL) was administered daily for the first month after transplantation, every other day for the second month, and every third day for the third month until euthanasia. In the OPCs group, DMSO was administered instead of ZJU-37.
All behavioral experiments were performed during the 11 weeks after transplantation of eight rats each group.
Morris water maze (MWM) test
This test was performed as previously described . Briefly, each rat entered the water training from four different quadrants and underwent two training sessions per day for five consecutive days. The latency to escape the water maze (the time from the rats entering the water to standing on the platform) was counted for each trial. On day 6, a probe test was performed by removing the platform and allowing each rat to swim freely for 60 s after entering the water from the first quadrant. The number of platform crossings and number and time that rats crossed through the platform quadrant were recorded. All data were recorded with a computerized video system.
Limb-use asymmetry test (cylinder)
The forelimb-use asymmetry test was used to assess sensorimotor function and behavioral symmetry. Rats were placed in a transparent Plexiglas cylinder (40 cm high, 20 cm diameter)  and initial forepaw (left/right/both) preference for weight-bearing contacts was scored. The forelimb asymmetryscore was calculated as: (right-left)/total of number of contacts.
Adhesive removal test
Sensory and motor functions were measured as described previously . All rats were familiarized with the testing environment for three days in a Perspex box. Two adhesive tapes were placed with equal pressu recovering the hairless parts on both forelimbs. The time to remove the adhesive tapes from each forelimb was recorded within a maximum of 120 s.
Rats from each group were deeply anesthetized with chloral hydrate at 12 weeks after transplantation and intracardially perfused with Phosphate-buffered saline(PBS) followed by fixation with 4% cold paraformaldehyde. Brains were dissected and post-fixed in the same solution for 12 h at 4°C, then sequentially dehydrated in sucrose (15% and 30%) until permeated. Coronal sections (14 µm thickenss) were cut on a freezing microtome (Leica Microsystems, Nussloch, Journal Pre-proof Germany) from the bregma anterior-posterior coordinates +1.0 to -1.0, collected on 3-aminopropyltriethoxysilane-coated slides (Sigma, St. Louis, MO, USA), and stored at -80°C in cryoprotectant solution. For protein analyses, fresh corpus callosum from sacrificed rats (4 from each sub-group) were isolated and stored at -80°C.
OPC apoptosis was assessed by TUNEL staining via a Dead End Fluorometric TUNEL System (Roche, Switzerland). Brain tissues were incubated overnight at 37℃ with the anti-PDGFRα (1:800) antibody. A standard TUNEL procedure was performed as described previously  after probing with a relevant secondary antibody. Nuclei were stained using 4', 6-diamidino-2-phenylindole (DAPI, Beyotime Biotechnology, Shanghai, China). TUNEL-positive cells were counted at five randomly chosen microscopic fields. OPC apoptotic rate was calculated as TUNEL and PDGFRα double-positive cells/total PDGFRα-positive cells per field × 100%.
Immunofluorescence staining was performed on the above-described sections of brain tissue (20 µm). Sections were blocked for 1 h in PBS containing 5% bovine serum albumin and 0.3% Triton X-100 at room temperature then incubated overnight with primary antibody. The following primary antibodies were used: anti-STEM121 (IgG, 1:500, TaKaRa), anti-myelin basic protein (MBP, IgG, 1:1000, Abcam), anti-glial fibrillary acidic protein antibody (GFAP, IgG, 1:500, Santa Cruz), anti-ionized calcium binding adaptor molecule-1 (Iba-1, IgG, 1:1000, Wako) and anti-NLRP3 (IgG, 1:500, Novus Biologicals). After washing three times with PBS, the samples were incubated with goat anti-mouse or goat anti-fluorescein isothiocyanate (FITC, IgG, 1:200, Santa Cruz) for 2 h at room temperature. Nuclei were stained with DAPI according to the manufacturer's instructions. Staining specificity was assessed by omitting the primary antibody. The number of GFAP-positive and Iba-1-positive cells was counted at three sections per rat from the same levels. Quantitative analysis of immunofluorescence staining was measured by Image J software(NIH, Bethesda, MD, USA).
Transmission electron microscopy (TEM)
Samples were prepared for electron microscopy according to previous protocols. In brief, brains were removed quickly after perfusion with 2% PFA/2.5% glutaraldehyde. The corpus callosum corresponding to the transplantation site (n=4 per group) were dissected and placed in 2.5% glutaraldehyde at 4°C overnight for post-fixation. After transferring to osmium tetroxide for 1 h at room temperature and dehydrating with increasing ethanol concentrations, the tissues were embedded in epoxy resin embedding medium. Ultrathin sections (50 nm) were made from the resin-embedded samples and observed under a transmission electron microscope. A total of 100 root axonal fibers from three samples in each group were measured at a magnification of 15,000 × g. Following image acquisition, axon and myelin diameters were measured using Image J software. The g-ratio, which is a structural index of remyelination and defined as the ratio of the inner axonal diameter to the total outer fiber diameter and lower ratios indicate more extensive myelination, was subsequently assessed. The average scores of ultrastructural myelin damage were determined as described previously.
Primary cell culture and drug treatment
Extraction and culturing of primary rat astrocytes and OPCs from newborn 0-2 day-old rat cerebral cortices were performed as previously described . Digestions were stopped with DMEM/F12 (1:1, HyClone) containing 10% fetal bovine serum (FBS, Clark Bioscience, Richmond). Cells were centrifuged at 1200 × g for 5 min, and supernatant was discarded. The medium was changed once every 2–3 days. After 9–11 days, microglia were dislodged using an orbital shaker (200× g for 1 h, 37°C), and OPCs were harvested by collecting the cell suspension after shaking the on ahorizontal orbital shaker for 18 h at 200× g and 37℃. The cells were then digested with 0.25% trypsin and seeded into 24-well plates at an appropriate density; the remaining adherent cells were astrocytes. Cells after the third generation were used for experiments and were divided into normal (Ctrl) and oxygen and glucose deprivation (OGD) groups. OGD-treated astrocytes were further divided into OGD, OGD-ZJU-37(5 µM) and OGD-ZJU-37(10 µM) groups and incubated in OGD-DMEM for 6 h. Finally, cells were collected for cellular immunofluorescence staining and western blot analysis. The astrocyte supernatant was mixed with DMEM/F12 at a ratio of 1:1. After oligodendrocytes grew for 2 days in DMEM/F12 containing 10% FBS, the mixed medium without OGD-astrocyte was used for the control (Ctrl) group and the medium with OGD-astrocyte with DMSO or ZJU-37 were used for the conditional medium-vehicle (CM-vehicle) group, the CM-ZJU37 (5μM) group and the CM-ZJU37 (10μM) group respectively for 24 h. Finally, the OPCs were collected for cellular immunofluorescence staining of PDGFRα and MBP and the Annexin V-FITC/PI to detect apoptosis by flow cytometry, separately.
Western blot analysis
Western blotting was performed as previously described . Total protein was extracted from the corpus callosum of the WMI rats and primary rat astrocytes were lysed with a lysis buffer and homogenized and centrifuged at 12,000 × g for 15 min at 4℃. The supernatants were collected and used for protein detection. Samples were run on a 10% SDS-PAGE gel and transferred tonitrocellulose membranes. Primary antibodies were: anti-NLRP3 (IgG, 1:500, Novus Biologicals), anti-ASC (IgG, 1:500, Santa Cruz), anti-caspase-1 (IgG, 1:500, Santa Cruz), anti-cleaved caspase-1 p20 (IgG, 1:1000, Cell Signaling Technology) and anti β-actin (IgG, 1:1000, Santa Cruz). The gray value of every band was analyzed with Image J software and reported as relative optical density of the specific proteins.
Three independent replicates were conducted for each experiment. Experimental data were analyzed with GraphPad Prism® software. After variation similarity was compared, one-way analysis of variation (ANOVA) followed by either the Newman-Keuls or Tukey honestly significant difference post hoc test was used for comparisons among multiple groups. A two-way ANOVA was used for escape latency in the Morris water maze training task. Quantitative data are expressed as mean±standard error of mean. Statistical significance was set at p < 0.05 for all tests.