Human (h)OPC culture and CM-Dil labeling
Human fetal brain tissue was derived from embryos aged 10–13 weeks, and cells were prepared in the Pediatric Laboratory of the Sixth Medical Center of the General Hospital of the Chinese People's Liberation Army (Beijing, China). Human fetal brain was mechanically dispersed, centrifuged, and separated to form a single-cell suspension, which was seeded into medium containing a cocktail of basic fibroblast growth factor, platelet-derived growth factor (PDGF), and neurotrophic factor-3 for the passage and amplification of neural stem cells [10]. Neural stem cells were induced to form OPCs to obtain relatively pure hOPCs [11]. Cells were cultured in a humidified atmosphere of 5% CO2 and 95% air at 37°C, and hOPC purity was evaluated by immunofluorescence staining with specific markers [ganglioside (A2B5), neural/glial antigen 2 (NG2), PDGF receptor-α (PDGFR-α), SRY-box transcription factor 10 (SOX10), oligodendrocyte transcription factor 2 (Olig2), O1, glial fibrillary acidic protein (GFAP), and class III β-tubulin (Tuj-1)].
To better trace and observe the transplanted cells, hOPCs were labeled with Cell Tracker CM-Dil (40718ES50; YEASEN, Shanghai, China) prior to transplantation by incubating the cells with the dye for 5 min at 37°C and others for 15 min at 4°C. After labeling, the cells were washed with PBS and resuspended in hOPC medium [10]. OPC medium was prepared by adding 2% B27 supplement, 20 ng/mL basic fibroblast growth factor, 1% penicillin/streptomycin, 5 µg/mL heparin, and 2 mM L-glutamine to Neurobasal-A medium. The labeling efficiency of the cells was verified at >95% prior to all transplantations by fluorescence microscopy (OLYMPUS, Tokyo, Japan).
Establishment of the WMI model and intranasal delivery of hOPCs
All experimental procedures were approved by the Animal Ethical and Welfare Committee of the Sixth Medical Center of Chinese People's Liberation Army General Hospital (Beijing, China). Pregnant Sprague–Dawley rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). At postnatal day 3 (p3), Sprague–Dawley rat pups (7–9 g) were anesthetized with 4% chloral hydrate and underwent hypoxia–ischemia (HI) by permanent right common carotid artery occlusion, followed by exposure to 6% oxygen and 94% nitrogen at 37°C for 90 min before being returned to their dam. In the sham group, only the right common carotid artery was exposed without common carotid cutting and subsequent hypoxia. For intranasal delivery, rats were administered 100 U hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA) dissolved in sterile PBS 30 min before administration of the cells. A Hamilton microsyringe needle (Hamilton Co., Reno, NV, USA) was inserted into the plastic hose of the drip liquid, followed by intranasal delivery of 6-µL drops (24 μL total) of cell suspension (1 × 106 cells) twice to each nostril. Droplets were administered into alternating nostrils at 15-min intervals. During these procedures, the mouth and opposite nostrils were closed to allow the cell suspension to pass into the nasal cavity of the animal. At 3 days after HI, experimental animals were randomly assigned to three different experimental groups: sham group, preterm white matter injury (PWMI) group and nasal transplantation group (n=3). Eight days after HI, experimental animals were randomly assigned to three different experimental groups: sham group, preterm white matter injury group and nasal transplantation group (n=10). All rats were intraperitoneally injected with cyclosporine (10 mg/kg/day) 3 days before transplantation.
Immunohistochemical staining
Animals were transcardially perfused with 4% paraformaldehyde solution at 12 weeks after hOPC application and post-fixed in the same solution for 24 h, followed by cryoprotection in 30% sucrose for 72 h. The brain was perfused to obtain frozen sections (thickness: 8 μm). After rewarming, the cells were washed three times in PBS for 5 min each. Coronal sections (8 μm) were successively blocked in 0.3% Triton X-100 and 5% bovine serum for 1 h at 37°C, followed by incubation with a primary antibody specific for myelin basic protein (MBP; 1:100, rat monoclonal; Abcam, Cambridge, UK) at 4°C overnight followed by secondary antibodies conjugated with Alexa Fluor 488 (1:200; Molecular Probes, Eugene, OR, USA) for 2 h at 37°C. The slides were washed again with PBS before mounting with DAPI.
Transmission electron microscopy and g-ratio analysis
Right hemispheres were used for transmission electron microscopy (TEM) at 12 weeks post-transplantation. Brain sections (1–3-mm thick) from the right corpus callosum of rats (n = 3/group) were fixed in 2.5% glutaraldehyde and 2% formaldehyde at 4°C for 72 h and then stained with osmium tetroxide overnight. The sections were dehydrated in an ethanol series and embedded in epoxy resin. Ultrathin sections were observed by TEM (Hitachi, Tokyo, Japan) after electron staining, and images were captured for further analysis. We selected 100 myelin sheaths (6000× visual field) from each sample to evaluate the myelin thickness (g-ratio) and classification, except in extremely long or distorted axons. Corpus callosum g-ratios were determined from the TEM images by manually measuring the axon diameter and outer myelin-layer diameter (axon average diameter/fiber average diameter) using Image-Pro Plus software (v.6.0; Media Cybernetics, Rockville, MD, USA).
Behavioral tests
Behavioral tests such as the Morris water maze test, adhesive-removal test, cylinder test, and gait analysis are described below.
Morris water maze test
Spatial learning and memory were evaluated at 12 weeks after OPC transplantation using the Morris water maze. An open circular pool [100 cm (diameter) × 50 cm (height)] was filled with water (24 ± 2°C), and the black platform [6 cm (diameter) × 28 cm (height)] was submerged 2–3 cm below the water surface at a depth of 30 cm. For the navigation trial, rats underwent four trials daily with different starting points over 5 consecutive days and twice daily. Rats escaping the platform were restricted within 60 s and allowed to rest on the platform for 10 s. The space-probe trial with the platform removed took place on day 6 over 60 s. The escape latency, number of platform crossings, number of platform quadrant crossings, and time spent on the platform quadrant were recorded.
Adhesive-removal test
The adhesive-removal test was used as a sensitive method for detecting and monitoring sensorimotor deficits after CNS injury. Before each test, the animals were placed in an experimental home cage for 2 min for acclimation. After testing, each rat was sterilized with alcohol. Prior to formal testing, animals were trained for 3 days to familiarize them with the test and exclude any outliers from subsequent testing. The longest detection time for each rat was 180 s. When the sticker was not removed after 180 s, the time was recorded as 180 s. The rats were gently scuffed and inverted, with a small quarter-circle adhesive placed on either the left or right forepaw. The time of contact and removal of the dot was measured separately, with the adhesive placed on alternating forepaws. The test was repeated three times for both the right and left forepaws.
Cylinder test
To evaluate forelimb-use asymmetry resulting from ipsilateral brain damage, the ratio of contralateral forelimb use was analyzed at 12 weeks post-transplantation. Each animal was individually placed in a glass cylinder [20 cm (diameter) × 40 cm (height)], and forelimb use was observed during the exploratory behavior of the rats for 5 min. The evaluation process was blind, and all mice were adaptively trained before starting the experiment. During the experiment, the environment was kept quiet to avoid unnecessary stimulation. Contact between the weight-bearing forepaw and cylinder wall during a full rear was recorded as right (normal), left (impaired), or both. Left forelimb preference was calculated as [(impaired forepaw + 1/2both)/(normal forepaw + impaired forepaw + both) × 100%].
Gait analysis
Gait analysis was performed at 12 weeks post-PWMI using an automated computer-assisted method (Xin Hai Hua Yi Instrument Co., Beijing, China), and data were collected and analyzed using Gait Analysis Lab software (v.5.0). An enclosed glass walkway was illuminated from the long edge with a green light that was completely internally reflected. The light reflected by the paws as they touched the glass floor was captured and transformed into a digital image. Food pellets were placed in the goal box as a motivator cue for a successful run. Training and experimental procedures were performed under the same conditions. Training started 1 week before surgery and lasted for at least 3 days until the animals learned to cross the walkway without interruption. An observer blinded to the experimental groups performed behavioral and data analyses. The data were then blindly assessed by trained technicians.
Statistical analysis
Data are presented as the mean ± standard deviation and mean ± standard error of the mean. Statistical analysis was performed using GraphPad Prism software (v.5.0; GraphPad, Inc. San Diego, CA, USA). The results of the water maze space-exploration experiment were evaluated by two-way analysis of variance, followed by multiple comparisons. Other results were analyzed by one-way analysis of variance. A p < 0.05 was considered to indicate significant results.