Animals and Experimental groups
Pregnant SD dams were obtained from the Experimental Animal Center of Qingdao, China. A total of 120 postnatal days (P) 3 rat pups, weighing 7.5–11.3g, were housed in temperature- and humidity-controlled cages with their moms in a 12-hour light/dark cycles. The experimental protocol was revised and 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 groups: sham 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), GroupD(n=24) and Group E (n=24).
Induction of WMD and Xenon treatment
As we have done previously[19] ,Pups were either administered lipopolysaccharide (LPS, 0.05 mg/kg, Escherichia coli 0111:B4; Sigma, USA) or normal saline (NS, Sham group) via intraperitoneal (i.p) injection. To avoid LPS-induced body temperature changes, pups were housed with their mothers after LPS or NS injection in an incubator for 3 hours to maintain their body temperature at 33 to 34°C before HI.[20] Next, pups were anesthetized with 5% chloral hydrate (0.01 ml/gm body weight, i.p) and HI was induced as described previously.[21] Briefly, the right common carotid artery (CCA) was exposed and separated from nerves and veins. Subsequently, the CCA was permanently ligated with 4-0 surgical silk, the wound was sutured and pups were returned to their cages to recover from anesthesia. The entire surgical procedure never exceeded 5 minutes. Sham-operated pups underwent the same operative procedure except for the ligation of CCA. After 1 hour recovery, pups in Groups B and C were placed in an airtight 3 L container partially submerged in a 36°C water bath, and exposed to humidified 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. Animals in group C, D and E were placed in a separate closed circuit container with 50% xenon (mixed with 30% oxygen and 20% nitrogen) for 3 hours.Following HI, the onset of the xenon treatment was delayed for 0, 2 hours, and 5 hours in groups C,D and E, respectively. Carbon dioxide was removed from the container by soda lime pellets. Pups were decapitated at 0, 24, 48 and 72 hours following xenon treatment, and control pups from groups A and B were simultaneously decapitated.
Hematoxylin and eosin staining
Brains were excised as described previously.[19] 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, Center Valley, PA,USA).
TUNEL staining
The apoptotic cells were detected with terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling assay(TUNEL,Roche) according to the instructions. 10 visual fields were randomly selected to count the number of positive cells, and the average value was obtained under a light microscope (OLYMPUS BX41, Olympus, Center Valley, PA, USA).
Western blot analysis
Brains were quickly extracted and the periventricular tissues were dissected and stored in aliquots at -80°C until further analysis. Periventricular brain tissues were homogenized in a cold lysis buffer supplemented with protease inhibitors and protein concentrations were determined using a BCA protein assay kit (Elabscience, Wuhan, China). Protein samples (40μg) were separated using 10% SDS-PAGE and blotted onto polyvinylidene fluoride membranes overnight at 4°C. Membranes were incubated with the appropriate primary antibody including anti-HIF-1α (1:500; Elabscience, Wuhan,China) and anti-GAPDH (1:1,000; Elabscience, Wuhan, China) overnight at 4°C. Next, membranes were incubated with anti-rabbit IgG antibodies conjugated to horseradish peroxidase (HRP; 1:50,000; Elabscience, Wuhan, China)and proteins were visualized by enhanced chemiluminescence. Band signals were quantified using 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 MixKit(Vazyme Biotech, Nanjing, China). Then using 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 min, 50˚C for 15 min ,85˚C for 5 min and 4˚C 10 min. 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.