Animals and WMI modeling
P5 SD rats (average weight 10–15 g) were purchased from Sichuan Dashuo Animal Science and Technology Co., Ltd (Chengdu, China). The rats were randomly divided into four groups: a Sham-operated group (Sham); a WMI model group (WMI); a group receiving Act A / phosphate-buffered saline (PBS) treatment after 24 h of WMI modeling (Act A/ PBS); and a group receiving Id2 overexpression lentiviral vector (Id2) and its corresponding vehicle(V), 6 h after Act A treatment (Id2/V).
The WMI model was established using the following procedure (Back, 2017): First, P5 neonatal rats were fixed on their backs after general anesthesia. The neck skin was then longitudinally incised for a length of about 1cm, and the right carotid artery was exposed and ligated after separation from glands and muscle tissue. After surgery, the rats were returned into an incubator for 30 min to recover. Then, they were placed in an 8%-oxygen and 92%-nitrogen cabin (8% O2 and 92% N2) with a gas flow rate of 3 L/min for 2 h to induce WMI. Rats were kept on a heating pad during surgical procedures to maintain the body temperature at 36–37 oC. The rats of the Sham group were only subjected to neck incision for dissociating the right carotid artery, without ligation or hypoxia. Following surgery, all neonatal rat pups were returned to their cages.
To establish the Act A/PBS group, rats were injected with 5ul of Act A (12.5 mg/kg, 25 mg/kg, 50 mg/kg)/PBS after 24 h of WMI induction using a Hamilton syringe needle (Hamilton, USA) via the lateral ventricle (LV), located 2 mm posterior and 2 mm lateral (right) from the bregma with a 2 mm needle depth. 4 ul of Id2 overexpression lentiviral vector/vehicle was injected via the LV after 6 h of Act A treatment to establish the Id2/V group.
Hematoxylin & eosin staining
At P7, rats were sequentially perfused with 0.9% normal saline and 4% paraformaldehyde (100 mL each), after which the tissues were extracted and post-fixed in a 4% paraformaldehyde solution for 24–36 h at 4 oC. Then, the tissues were paraffin-embedded and serial sectioned in coronal position for 5mm, and three sections containing corpus callosum (CC) (0.26mm-1.80mm behind the anterior fontanelle according to the mapping of rat brain) were selected for analysis. Finally, the sectioned tissues were stained with hematoxylin & eosin (HE) and observed using a Leica inverted optical microscope (Leica, Germany). In each animal, four randomly selected fields were examined. Six animals per group were analyzed.
Brains were taken at P7, P14, P21, P28, and P35 and post-fixed in 4% paraformaldehyde at 4oC for at least 48 h, then embedded in 2–3% agarose. Coronal brain sections (thickness 40 µm) were cut using an oscillating tissue slicer (Leica, Germany). Three sections containing CC (0.26 mm-1.80 mm behind the anterior fontanelle according to the mapping of rat brain) were selected for analysis. The sections were first washed in PBS and incubated in 0.3% Triton X-100 at room temperature for half an hour, and then incubated for 1 h in fetal calf serum to inhibit non-specific binding. Second, the brain sections were incubated with the primary antibodies (rabbit anti-Id2 polyclonal antibody,1:500, novusbio; rabbit anti-BMP4 polyclonal antibody, 1:1000, abcam; rabbit anti-Olig2 polyclonal antibody, 1:500, Millipore; rabbit anti-Ki67 polyclonal antibody, 1:500, abcam; mouse anti-Vimentin monoclonal antibody, 1:200, abcam; rabbit anti-CC3 polyclonal antibody, 1:500, Cell signaling; rabbit anti-NG2 monoclonal antibody, 1:200, Proteintech; rabbit anti-O4 monoclonal antibody, 1:25, Millipore; mouse anti-APC antibody [CC-1], 1:200, abcam; mouse anti-MBP monoclonal antibody, 1:1000, arigo; rabbit anti-MAG monoclonal antibody, 1:100, CST; rabbit anti-PLP polyclonal antibody, 1:1000, abcam; mouse anti-Tau1 monoclonal antibody, 1:1000, Millipore; mouse anti-SMI31 monoclonal antibody, 1:1000, biolegend; mouse anti-SMI312 monoclonal antibody,1:1000, biolegend) at 4 oC overnight, then incubated for 2 h at room temperature with secondary antibodies which were Cy3-conjugated donkey anti-rabbit IgG, Cy3-conjugated donkey anti-mouse IgG, 488-conjugated donkey anti-rabbit IgG, 488-conjugated donkey anti-mouse IgG. The sections were mounted onto glass slides with Fluorescent Mounting Medium (Beyotime, China). Finally, fluorescence imaging was performed using a confocal laser scanning microscope (Olympus, Japan) and FV-ASW-3.1 software (Olympus). The mean fluorescence intensity was defined as the ratio between the sum of the integral optical density of the target protein and the area. Positive cells and mean fluorescence intensity counting were performed for each field with a 40X objective lens (field size, 0.24 mm2), using the Image J software. In each animal, four randomly selected fields from the CC were examined. Six animals per group were analyzed.
The isolated CC was treated with a brain tissue protein extraction kit (Chengdu beibokit, BB-31227-1). Lysates were centrifuged at 14,000 rpm for 30 min at 4 °C. The protein concentration was determined through a BCA protein assay kit (Pierce) using bovine serum albumin (BSA) as the standard. Protein samples were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels. The protein was then transferred to polyvinylidene difluoride (PVDF) membranes, which were blocked in 5% non-fat dry milk in TBS containing 0.05% Tween 20 for 1 h at room temperature, with rotation. The membranes were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-Act A polyclonal antibody (1:500, novusbio), mouse anti-MBP monoclonal antibody (1:500, arigo), rabbit anti-MAG monoclonal antibody (1:100, CST), rabbit anti-PLP polyclonal antibody (1:500, abcam), mouse anti-Tau1 monoclonal antibody (1:500, Millipore), mouse anti-SMI31 monoclonal antibody (1:500, biolegend), mouse anti-SMI312 monoclonal antibody (1:500, biolegend), mouse anti-Noggin monoclonal antibody (Abcam,1:200), rabbit anti-BMP4 polyclonal antibody (1:500, abcam), rabbit anti-Id2 polyclonal antibody (1:500, novusbio), and a mouse anti-actin polyclonal antibody (Santa Cruz Biotechnology, 1: 5000) was detected as the loading control. Following washes, the membranes were incubated with peroxidase conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Santa Cruz Biotechnology, 1: 5000) in blocking solution for 1 h. The bound antibodies signals were developed by enhanced chemiluminescence (Pierce, Rockford, IL). The immunoreactivity of the signal bands was quantified using the Image J software. The relative expression level of the target protein was calculated as the target protein integrated density values (IDVs) relative to actin IDVs. All experiments were repeated at least three times to ensure the reproducibility of the results.
Rats were sequentially perfused with 0.9% normal saline and 4% paraformaldehyde (100 ml each) at P35. The rat brains were taken and sectioned to the size of approximately 1 mm3 including the CC at 1.2 mm to 3.0 mm posterior to the bregma. The sectioned tissue was pre-fixed with a mixed solution of 3% glutaraldehyde. Then post-fixed in 1% osmium tetroxide, dehydrated in an acetone series, filtrated in Epox 812, and embedded. Next, the semi-thin sections were stained with methylene blue, the ultrathin sections were stained with uranyl acetate and lead citrate. Finally, the ultrathin sections were examined with a transmission electron microscope (EM) (H-600IV; Hitachi, Japan). Myelinated axons were counted for each field using the Image Pro Plus 6.0 software. In each animal, four randomly selected fields from the CC were examined. Six animals per group were analyzed.
Morris water maze
Behavioral testing using the Morris water maze (MWM) was performed from P29 to P35. The testing facilities includes a circular tank (1.5 m in diameter) and a location-constant platform (14 cm in diameter) placed 1.5 cm under the surface of the water. The water temperature was maintained at 25±1 oC during testing. The test consists of two parts, namely place navigation training and space exploration, both of which are aimed to test spatial learning and memory ability.
The place navigation training was conducted during the first 6 days (P29–P34), For which the rats were trained to swim in the four alternating quadrants. From each quadrant, the rats swim in the water for 120 s. If the platform is successfully found during this period, the escape latency is recorded as the time at which the rats find the platform. If the rats fail to find the platform within 120 s, it is guided to it by a researcher and stays on the platform for 30 s, and the escape latency time being recorded as 120 s. The time at which the rat found the platform in each training session was recorded, and the average of the four quadrant latency periods was computed as a daily final score representing the ability to acquire the spatial information.
The platform was removed and the space navigation test was conducted at P35 to test the memory retention ability of the rats, 24 h after the last place navigation training. The rats were free to swim in the tank for 120 s from the third quadrant starting point. The trials were recorded using a video camera on the ceiling, and the platform crossing time was calculated and analyzed using the tracking system (Mengtai, China).
Quantification analysis and statistics
All images were acquired from the same CC area. All data were presented as mean ± standard deviation (SD). All graphs were produced using GraphPadPrism 8.0. A Student's t-test was used when comparing between two groups. Analysis of variance (ANOVA) was used when comparing more than two groups, followed by the Student’s t-test if homogeneity of variance was assumed or by Dunnett’s test if homogeneity of variance was not assumed. All statistical analyses were performed using SPSS 23.0. P-values *P <0.05, or **P < 0.01 were considered statistically significant.