The study selected P7 C57BL/6 pups (male or female), weighing 3 to 5 g, provided by the Guangdong Medical Laboratory Animal Center (Guangzhou, Guandong). Each squirrel cage allows one female mouse to take care of its pups. Mice are reared in an environment with a temperature of 20 to 24°C and a humidity of 40% to 70%. All animal-related experiments done in this study have been approved by the Experimental Laboratory Animal Committee of Guangdong Pharmaceutical University and comply with the Chinese Council on Animal Care guidelines.
HIBD model and macamide B administration
In this experiment, we used a total of 160 P7 C57BL/6 pups. The improved Rice-Vannucci method was used to construct the HIBD model [31, 32]. Briefly, C57BL/6 pups at 7-9 days of age (P7-9) were given continuous inhalation anaesthesia (isoflurane) through a face mask, fixed on a sterile surgical drape in the supine position, and disinfected at the predetermined surgical incision location on the neck skin. A 1 cm incision was cut along the middle of the neck, the subcutaneous tissue was bluntly separated, and the unilateral common carotid artery (CCA) was separated, a coagulator was used to cut off the CCA, the skin was sutured and disinfected, and the pups were returned to the dams for feeding and recovery. After recovering for 1 h, put the pups in a 37°C water bath controlled hypoxia box, and a mixed gas of oxygen (8%) and nitrogen (92%) was supplied. The pups were exposed to hypoxia for 4 h then returned to the dams to feed, and the model was completed.
Macamide B (Shanghai yuanye Bio-Technology Co., Ltd, China, HPLC≥98%) was dissolved in PBS solution (1.25 mg/ml). Twenty minutes before the ischemia surgery, the macamide B group mice were intraperitoneally injected with macamide B (60 mg/kg). For pups in the vehicle group, give the same volume of PBS treatment. The protocol is described in Fig. 1c.
Infarct volume measurement
After HIBD for 24 h, intraperitoneal administration of 10% chloral hydrate to the pups for anaesthesia (0.1 ml), and the brains were immediately extracted. We cut the pup's brain into four brain slices in a coronal plane (the intervals between adjacent brain slices is 2 mm). The sections were stained for 20 min in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich, Germany), turning the brain slices from time to time to make even contact with the staining solution. Then the images of the brain slice of the pup were captured by a digital camera. The nonischemic necrotic area was red, while the ischemic necrotic tissue was white. Use ImageJ software (version 1.8.0, USA) to analyze the cerebral infarct volume of pups. The percentage of infarct volume = [(contralateral hemisphere − un-infarcted area of ipsilateral hemisphere)/contralateral hemisphere × 2] × 100% .
Measurement of brain water content
After HIBD for 24 h, intraperitoneal administration of 10% chloral hydrate to the pups for anaesthesia (0.1 ml), and the brains were immediately extracted. The pup's brain was divided into two parts and weighed for wet weight and the cerebral hemispheres were dried in an oven at 106°C for 24 h and weighed for dry weight. Percentage of brain water content = (wet weight–dry weight)/wet weight ×100%. The percentage of brain water content = (wet weight - dry weight) / wet weight × 100%.
Neurological damage caused by HIBD can lead to sensorimotor impairments. Body weight and sensorimotor performance (righting reflex, negative geotaxis, and grip test) were tested 1, 3, and 7 days after the HI procedure in a blinded manner .
The righting reflex is used to evaluate the recovery of the brain of pups. The pups were placed on a flat surface with one hand gently holding the head and the other hand gently holding the hind limbs; the pup was rolled onto its back and released. The time required for the mouse to return to an upright position (all limbs on the ground) was recorded. The maximum testing time was 1 min, and times over 1 min were recorded as 1 min.
The geotaxis test was used to diagnose vestibular or proprioceptive functions. Place the pup on a flat surface inclined at 45 degrees, with the head of the pup facing the bottom of the plane, and record the time it takes for the head and body of the pup to make a 180° turn. The maximum testing time was 1 min, and times over 1 min were recorded as 1 min.
The pup grasped a metal wire with a diameter of 1.5 mm with both front feet. The metal wire was stretched horizontally in a test box with a width of 50 cm. The distance between the metal wire and the bottom of the box was 15 cm. The bottom was covered with cork chips. Record the time it takes for the pup to grasp the wire to release it. The minimum testing time was 20 s, and times below 20 s were recorded as 20 s.
Western blot analysis
Protein samples (15 μl/well) were separated on SDS-PAGE gel (80 V, 110 min) and transferred to 0.22 μm polyvinylidene difluoride (PVDF) membrane (290 mA, 100 min). Five percent nonfat dry milk in TBST buffer was used to block nonspecific sites for 1.5 h. Incubate the primary antibody in a refrigerator at 4℃ for 16 h. Use that following primary anti-antibody: PI3K (1:1500, Abcam, UK), p-PI3K (1:1500, Abcam, UK), AKT (1:1500, Proteintech, USA), p-Akt (1:1500, Cell Signaling Technology, USA), Beclin1 (1:750, Abcam, UK), LC3B (1;500, Proteintech, USA), p62 (1:1000, Abcam, UK), p53 (1:500, Multisciences, China), Bax (1:750, Proteintech, USA), Bcl-2 (1:750, Proteintech, USA), caspase-3 (1:600, Proteintech, USA). and cleaved caspase-3 (1:650, Proteintech, USA). β-actin (1:800; Proteintech, USA) was employed as an internal reference protein. After 16 h, the corresponding secondary antibodies (1:6000, EarthOx, USA) were incubated for 90 min. Bands were measured by an automatic chemiluminescence image analysis system (Tanon 5200, Shanghai, China). ImageJ software was used to carry out Western blot analysis, and SPSS 21.0 was applied to process the data.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining.
Because cells are prone to programmed cell death during HI . Therefore, we assessed the apoptotic cells in the brain tissue of the pups by TUNEL Apoptosis Detection Kit (fluorescence) (Wanleibio, China).
On day three after HI damage, the pups were perfused into the heart, and fresh brain tissue was quickly collected in 4% paraformaldehyde (PFA) for fixation. After fixation for 24 h, the brain tissue of the pup was rinsed under running water for 16h. After washing with running water, perform gradient dehydration, paraffin embedding, tissue sectioning and other operations on the brain tissue of pups for subsequent TUNEL detection.
The slices were immersed in xylene Ⅰ and Ⅱ for 15 min each, immersed in 100% Ⅰ, 100% Ⅱ, and 95%, 90%, 80%, 70%, and 50% ethanol solutions for 5 min per solution, and washed with ddH2O for 3×5 min. Put that slices into citrate buffer solution (0. 01 mol/L), and place them in a microwave oven at high-temperature repair for 20 min. Add 25 μl of 3% H2O2 buffer to each brain tissue and let stand for 12 min in the dark, followed by PBS washing for 3×5 min. Then, add 25 μl TUNEL reaction buffer to each brain tissue and incubated in a 37°C incubator in the dark for 90 min. After washing with PBS for 3×5 min, counter-stain brain tissue cell nuclei with DAPI for 5 min, then repeat the PBST wash step. After absorbing excess water with filter paper, add 25 μl of anti-fluorescence quencher to each brain tissue for mounting, immediately observe with a fluorescence microscope (Olympus BX51, Japan) , or store it at -20°C for observation within a week.
Fluoro-Jade C Staining
After HIBD for 24 h, the pups were immediately sacrificed, and brain paraffin sections were made. Fluoro‑Jade C (FJC) staining (Biosensis, USA) was used to detect the degeneration of neurons in the pup's brain tissue.
The slices were immersed in xylene Ⅰ and Ⅱ for 15 min each, immersed in 100% Ⅰ and 100% Ⅱ ethanol solutions for 5 min per solution, immersed in 70% ethanol solutions for 5 min per solution, and washed with ddH2O for 2×5 min. Transfer slides to a new Coplin jar containing ddH2O for 2 min. Mix 9 parts of ddH2O and 1 part of potassium permanganate solution and add to the brain tissue slices and incubate at room temperature for 10 min. Rinse slides for 2 min in ddH2O. Mix 9 parts of ddH2O, 1 part of FJC solution and 1 part of DAPI solution and add to the brain tissue slices and incubate for 12 min. Wash in ddH2O 3×1 min. Put the slices in a 56°C oven and bake for 5 min. The dry slides are then cleared by brief (5 min) immersion in xylene. Mount the slides with an anti-fluorescence quencher. Immediately observe with a fluorescence microscope, or store it at -20°C for observation within a week.
Tissue immunofluorescence staining
After HIBD for 24 h, the pups were immediately sacrificed, and brain paraffin sections were made. Tissue immunofluorescence staining was used to detect the expression levels of p53, Bax, Bcl-2, caspase-3, and cleaved caspase-3 in the brain tissue of mice.
Repeat tissue dewaxing and high-temperature repair steps, and incubate with Quick BlockTM immunostaining blocking solution for 20 min. Incubate each brain tissue section with the primary antibody at 4°C for 16 h, and add 0.5% Triton X-100 to the corresponding primary antibody for cell rupture. The following primary antibodies were used:Beclin1 (1:200, Abcam, UK), LC3B (1;250, Proteintech, USA), p62 (1:260, Abcam, UK), p53 (1:260, Multisciences, China), Bax (1:180, Proteintech, USA), Bcl-2 (1:260, Proteintech, USA), caspase-3 (1:280, Proteintech, USA), and cleaved caspase-3 (1:280, Proteintech, USA). After 16 h, the membranes were removed from the refrigerator, allowed to return to room temperature for 30 min, and washed again with PBST for 3×10 min. Dylight 488 labelled goat anti-rabbit fluorescent secondary antibody (1:360, Sigma-Aldrich, USA) or Dylight 594 labelled goat anti-rabbit fluorescent secondary antibody (1:360, Sigma-Aldrich, USA), incubate for 2 h at room temperature in the dark. After washing with PBS for 3×5 min, counter-stain brain tissue cell nuclei with DAPI for 5 min, then repeat the PBST wash step. Mount the slides with an anti-fluorescence quencher. Immediately observe with a fluorescence microscope, or store it at -20°C for observation within a week.
All of the experiments were repeated at least three times. The data are presented as the mean ± SEM. Statistical analyses were carried out by SPSS.21.0 and GraphPad Prism (version 8.0, USA). Differences between individual groups were first compared using analysis of variance (one-way ANOVA), and then post hoc testing was analyzed with Tukey or Student-Newman-Keuls multiple comparisons. Difference between the two groups were compared using Student's t-test. A p < 0. 05 indicated that the difference between the two groups was statistically significant.