Animals and Experimental groups
Experiments were performed with 120 SD rat pups (the Experimental Animal Center of Qingdao, China), postnatal days 3 (P3) rat pups, weighing 7.5–11.3g, housed in temperature- and humidity-controlled cages with their moms. Light was maintained in a 12-h light/dark cycle. All protocols were approved by the Institutional Animal Care and Use Committee of Qingdao University. All measures were taken to minimize animal discomfort.
Pups were randomly assigned into 3 different groups: sham-operated group (Group A, n=24), LPS + HI group (Group B, n=24,), and LPS + HI + Xe (n=72). According to the onset of xenon treatment, the LPS+HI+Xe group was further divided into three subgroups, Group C (n=24), Group D(n=24) and Group E (n=24). which are separately delayed 0 hour, 2 hours, and 5 hours after HI.
Induction of WMD and Xenon treatment
Rat pups in Group B and Group C were first injected intraperitoneally (Ip) with LPS (Escherichia coli 0111:B4; Sigma, USA), while the Sham-operated pups (Group A) injected with normal saline (NS). To avoid LPS-induced body temperature changes, the rat pups were returned to their dams after LPS or NS injection, and housed in an incubator to maintain body temperature at 33 to 34°C before HI for 3 hours [19]. Next, pups were anesthetized with 5% chloral hydrate (0.01 ml/gm body weight, i.p) and HI was induced as described previously. [20]. briefly the right common carotid artery (CCA) was exposed, separated from nerves and veins and permanently ligated with 4-0 surgical silk. After the wound was sutured, the pups were allowed to return to the cages and recover from anesthesia. The entire surgical procedure never exceeded 5 minutes. Sham-operated pups underwent the same operative procedure except that the exposed carotid artery was not ligated. After surgery, the pups were returned to the cages. After 1-hour recovery, pups in Group B and Group C, D, E were placed in an airtight 3 L container partially submerged in a 36°C water bath, and exposed to humidify 8% oxygen (a mixture of 8% O2 and 92% N2) with a flow rate of 3 L/minute for 1 hour. Following hypoxia, pups in Group B were returned to the cage until they were sacrificed. Group C, Group D, Group E were separately placed in a closed circuit container within 50% xenon (mixed with 30% oxygen and 20% nitrogen) for 3 hours[21]. The start of the 3 hours of xenon-treatment in Group C, Group D and Group E was separately delayed 0 hour, 2 hours, and 5 hours after HI. Carbon dioxide was removed from the container by soda lime pellets. The decapitation of rats was begun 0 hour, 24 hour, 48 hours and 72 hours after xenon treatment, and the rats in Group A and Group B were also decapitated at the same time with Group C. Animals were allowed to survive 3days, 4 days, 5 days and 6 days.
Hematoxylin and eosin staining
Brains were excised as described previously. [22] Briefly, pups were anesthetized with chloral hydrate and fixed on an operating panel. Next, the heart was exposed and a 10 ml syringe with 4% paraformaldehyde was inserted from the left ventricle to the aorta. Paraformaldehyde was injected into the heart until clear liquid flowed out from the atrium and the extremities became pale and stiff. Subsequently, the skull was systematically stripped to expose cerebral tissue. The periventricular brain tissue was dissected, immediately placed in 4% paraformaldehyde solution at room temperature for 48 hours, dehydrated in ethanol series, and embedded in paraffin. Paraffin blocks were coronally sectioned at a 10-μm thickness from the genu of the corpus callosum to the end of the dorsal hippocampus. Hematoxylin and eosin (HE) staining was performed in four sections per brain. Stained sections were observed under a light microscope (OLYMPUS BX41, Olympus, and Center Valley, PA, USA).
TUNEL staining
Apoptosis was detected with terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling assay (TUNEL, Roche) according to the manufacturer’s protocol. Cells showing brown nuclei were considered as TUNEL positive cells and the average number of TUNEL positive cells from 5 randomly selected areas was counted under a light microscope (OLYMPUS BX41, Olympus, Center Valley, PA, USA).
Western blot analysis
Brain tissues were stored in aliquots at -80°C until needed for further analysis. Western blotting was carried out to detect the levels of HIF-1α in the white matter. The Brain tissue samples were homogenized in cold lysis buffer and the protein concentrations were determined using a BCA Protein Assay kit (Elabscience, Wuhan, China). Samples (40μg) were separated using 10% SDS-PAGE and blotted onto polyvinylidene fluoride membranes overnight at 4°C. The membranes were incubated with primary antibodies including anti- HIF-1α (1:500; Elabscience, Wuhan, China), and anti-GAPDH (1:1,000; Elabscience, Wuhan, China). After incubation, membranes were washed 5 times for 3 minutes. Proteins were visualized using anti-rabbit IgG antibodies which were conjugated to horseradish peroxidase (HRP)(1:50,000;Elabscience, Wuhan, China). Immunoreactivity was detected by horseradish-conjugated secondary antibodies and visualized by enhanced chemiluminescence. The band signals were quantified using an imaging software (BandScan 5.0).
Real-Time Reverse Transcription–PCR
Total RNA was extracted from brain tissue using Trizol (Aidlab, Lot:252250AX, Beijing, China) followed by quantification and reverse transcription using HiScript Reverse Transcriptase (RNase H) reagent Kit (Gene Copoeia,MD, USA) following the standard protocols. Next, real-time PCR was performed using the2×All-in-one tmqPCR Mix Kit(Vazyme Biotech, Nanjing, China). Then used specific miR-210 and an endogenous control U6 stem‑loop primer, miR-210 or U6 stem-loop reverse transcriptase primers in a 20µl system buffered with RT buffer and Distilled De-ionized Water(ddH2O). The RT thermal cycle program was as follows: 25˚C for 5 mins, 50˚C for 15 mins, 85˚C for 5 mins and 4˚C 10 mins. The resulting cDNA was stored at -20˚C.qPCR reaction(40 cycles) at 50°C for 2 mins,at 95°C for 10mins,at 95°C 30 s,and at 60°C for 30 s.After amplificationg,the relative gene expressions were calculated in accordance with the ΔΔCt method. Relative miRNA levels were expressed as 2 -ΔΔCt and ratios to control. Samples were analyzed in triplicates and the data from 6 RT-PCR samples were averaged.
Statistical analysis
All data were expressed as mean + SE. Statistical analysis was performed using IBM SPSS Statistics. The data conformed to normal distribution (P > 0.05), and met the homogeneity of variance (P > 0.05). Variance analysis was used and LSD was used to compare the two. Data didn’t conform to normal distribution (P < 0.05), or to normal distribution but didn’t met the homogeneity of variance, non-parametric test was used and two comparisons were made, p < 0.05 was considered statistically significant.