Rats

The animals required for this study were purchased from Chengdu Dashuo Experimental Animal Co., LTD. (Sichuan, China), and all were 3–4 months old, including female SD rats weighing 300–350 g and male SD rats weighing 350–400 g. This experiment complied with the UK Animal Science Procedure guidelines and was performed in accordance with the UK Animals (Scientific Procedures) Act of 1986 and the National Research Council Guide for the Care and Use of Laboratory Animals. This study was also approved by the Animal Ethics Committee of Chengdu Medical College. This study has not been registered.

Male and female rats were randomized in a 2:1 ratio, allowed to eat and drink ad libitum, and acclimated in standard cages maintained at 22 ± 2 °C, a relative humidity of 50–60%, and a 12 h light–dark cycle for 1 week. As previous literature reported that sex has little effect our experiment, we did not distinguish between the sexes of the pups. Necessary precautions were taken to minimize pain and stress.

We included term male and female SD rat pups born between E21 and E22. To ensure the comparability of the two groups, we limited the number of pups to 11–13 pups per group. A total of 120 rats were included, of which 42 were assigned to the sham-operation group and 78 were assigned to the WMD model group. P0, the first time point after birth, is defined as no more than 6 h after birth. At P1–P5 (between days 1 and 5 after birth), rodent brains are equivalent to human brains at 23–32 weeks gestation. This time frame is therefore suitable for modeling brain injury in preterm infants [21]. Neonatal rat body weight was monitored at P0, P2, and P5, and brain tissues were randomly collected at different time points after birth (P2 and P5). Animals that died were excluded from the study. Adult rats continued to be fed or were used for other studies following the “3R” (replace, reduce, and refine) principle [22]. In this study, each group included six samples, and three independent replicates were performed for each sample.

Intracerebroventricular Injection

After stabilization of the intrauterine inflammation group in the normal environment for 6 h, 1 µL LPS (L2880-10 mg; Sigma, St. Louis, Missouri, USA) dissolved in 0.1 mg/mL saline (total dose 1 mg/kg) was stereotaxically injected into the ipsilateral hemisphere ventricle of rat pups (coordinates: 2 mm posterior, 1.5 mm transverse, 3 mm below the skull surface) at a flow rate of 0.5 µL/min [23, 24]. The LPS dose was determined from a concentration gradient established in a preliminary study. The body weight of neonatal rat was fixed at 6.0 ± 1.5 g, and the total dose of LPS was 6.0 ± 1.5 µg. After LPS injection, rats in the intervention group were injected with 1 µL SC79 (S7863, 20 µmol/L dissolved in dimethyl sulfoxide, diluted in normal saline according to the manufacturer’s instructions) or rapamycin (RAPA; S1039, 5 mM dissolved in dimethyl sulfoxide, diluted in normal saline according to the manufacturer’s instructions) at P2 in the lateral ventricle. Sham-operated rats were injected with the same volume of solvent. Brain tissue was sampled from the cerebral hemisphere contralateral to the ventricular injection site. To reduce pain, rats were deeply anesthetized with an intraperitoneal injection of 4% pentobarbital sodium (200 mg/kg body weight) prior to euthanasia. After surgery, all neonatal rats were numbered according to a random number table generated in Excel (Microsoft, Redmond, WA, USA). Random numbers were sorted from small to large and assigned to different groups. Numerical sample identifiers were used during experimental procedures and data analysis, and the researchers were blinded to the treatment.

Histopathology

Rat brain tissues were collected, embedded in paraffin, and sectioned for pathological examination. Briefly, animals were deeply anesthetized by intraperitoneal injection of 4% sodium pentobarbital (200 mg/kg body weight) and perfused with normal saline via the cardiac vein, followed by injection of 4% paraformaldehyde. Tissues were fixed in paraformaldehyde at 22 ± 2 °C for 72 h, dehydrated in gradient alcohol, embedded in paraffin, and sectioned in the coronal plane (5 μm thick). Tissues from the corpus callosum to the dorsal end of the hippocampus were stained with hematoxylin and eosin (H&E; Cat# G1121; Solarbio, Beijing, China). Randomly selected microscope fields were captured from six serial sections from each brain using a Leica microscope (DM4000B; Leica, Wetzlar, Germany) and 40x objective magnification and analyzed by a blinded investigator using ImageJ 1.8.0 (RRID: SCR_003070; National Institutes of Health [NIH], Bethesda, MD, USA).

Immunohistochemistry and Immunofluorescence

Myelin basic protein (MBP) and 2ʹ,3ʹ-cyclic nucleotide 3ʹ-phosphodiesterase (CNPase) expression levels were quantified by H&E, immunofluorescent, and immunohistochemical staining to determine oligodendrocyte maturity and evaluate the degree of WMD. For immunofluorescence and immunohistochemical analyses, tissues were blocked with 1× phosphate-buffered saline, 10% normal goat serum, and 0.1% Triton X-100 for 10 min. For immunohistochemical analysis, 3% hydrogen peroxide was added for 10 min to inhibit endogenous peroxidase activity. The sections were then incubated with a primary antibody solution containing goat anti-MBP (1:100; RRID: AB_305869; Abcam, Cambridge, UK), goat anti-CNPase (1:100; RRID: AB_2082593 Abcam), anti-Beclin 1 (1:100, Cat#66665-1-Ig, Proteintech, China), anti-Bcl-2 (1:100, Proteintech, RRID: AB_2227948), and OPC-specific O4 (1:100; Cat#MAB1326-SP; R&D Systems, Minneapolis, MN, USA) antibodies. Tissues were subsequently incubated with enzyme-conjugated goat anti-mouse immunoglobulin G (IgG) secondary antibody (Cat #SP-900; ZSGB-Bio, Beijing, China) for 15 min at 37 °C, followed by incubation with 3,3ʹ-diaminobenzidine working solution (Cat #ZLI-9019; ZSGB-Bio). MBP signal intensity was expressed as mean MBP fluorescence intensity. ImageJ 1.8.0 was used to determine the optical density of each image pixel. The integrated optical density (IOD) was measured, and the average optical density (AOD; IOD/target distribution area) was calculated. To evaluate the binding of Beclin 1 to Bcl-2 in O4+ cells, the antibodies for each were simultaneously used for labeling and observed under a laser confocal microscope (Nikon A1, ver.4.10).

For immunofluorescent staining, the sections were treated with an appropriate fluorescein isothiocyanate-conjugated secondary antibody (1:300; Cat #550,066; ZenBio, China).

Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling Assay

Paraffin-embedded sections were deparaffinized, rehydrated, and antigen-repaired in citrate buffer (pH 6.0) for 8 min and then incubated with 20 mg/mL proteinase K (Cat #ST533; Beyotime, Shanghai, China) dissolved in Tris/HCl for 30 min at 22 ± 2 °C. The sections were subsequently incubated in a terminal deoxynucleotidyl transferase (TdT) enzyme reaction solution (Cat# KGA700; KeyGen Biotech, Nanjing, China) for 1 h at 37 °C in a wet box. O4-antibody (1:100; Cat #MAB1326-SP; R&D Systems, Minneapolis, MN, USA) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL; Cat #G1501-100T; Servicebio, Wuhan, China) fluorescent double staining was used at P2 to determine whether SC79 can reduce excessive OPC apoptosis.

Western Blotting

Western blotting was performed using a dioctanoic acid protein detection kit (Cat #PC0020; Solarbio). Samples (40 to 60 µg/10 µL) were separated using 8% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyvinylidene difluoride membranes were subsequently used for protein transfer, and samples were incubated overnight at 4 °C with the following primary antibodies: anti-p-Akt (Ser473) (1:2000; RRID: AB_2315049; Cell Signaling Technology), anti-Akt (1:1000; RRID: AB_915783; Cell Signaling Technology), anti-p-mTOR (1:2000; Cat #R25033; ZenBio), anti-mTOR (1:2000; Cat #380,411; ZenBio), anti-p62 (1:2000; RRID: AB_10694431; Proteintech), anti-LC3 (1:1000; RRID: AB_2137737; Proteintech), anti-Bax (1:10 000; RRID: AB_2061561; Proteintech, Planegg-Martinsried Germany), anti-Bcl-2 (1:1000, Proteintech, RRID: AB_2227948), anti-cleaved caspase 3 (1:1000; Cat #341,034; ZenBio), anti-caspase 3 (1:1000; RRID: AB_331439; Cell Signaling Technology), anti-caspase 9 (1:500; RRID: AB_2068632; Proteintech), and anti-β-actin (1:5000; Cat #CL594-66009; Proteintech). Goat anti-mouse or anti-rabbit IgG secondary antibodies were incubated with horseradish peroxide (1:500; RRID: AB_449890; Abcam, Cambridge, UK). Signals were detected using a bioimaging system (ChemiDoc XRS+; Bio-Rad), and ImageJ software was used to analyze the band intensity.

Statistical Analysis

Data are presented as the mean ± standard error of the mean (SEM). Outlier testing was not performed due to the small sample size. The Shapiro–Wilk test was used to evaluate normality of the experimental data. An unpaired Student’s t test was used to compare the means of two groups, and one-way or two-way analysis of variance (ANOVA) was used to compare multiple groups. Prism 8.02 software (RRID: SCR_002798; GraphPad, San Diego, California, USA) was used for the statistical analyses. P < 0.05 was considered statistically significant.