The neuroprotective effects of xenon on neonatal rats with white matter damage by regulating expression of microRNA-210 and HIF-1α

Premature birth is a significant health care burden. White matter damage (WMD) is a leading cause of acute mortality and chronic morbidity in preterm birth. Xenon(Xe) is a general anesthetic with neuroprotective effects.We aimed to reveal the molecular basis and neuroprotective mechanism of Xe intervention in treating white matter damage by detecting the expression level of microRNA 210 (miR-210) and hypoxia inducible factor 1α (HIF-1α) in brain tissues of 3-day-old neonatal rats. Methods Three-day-old SD rats were randomly divided into sham group(Group A, n=24), lipopolysaccharide(LPS)+hypoxia-ischemia(HI) group (Group B, n=24) and LPS+HI+Xe group (n=72). The onset of Xe inhalation started at 0,2 and 5 hours in subgroups C,D,and E respectively.We investigated the neurobehavioral deficits by performing TUNEL and hematoxylin and eosin (HE) staining and examining the expression of miR-210and HIF-1α in brain tissues via RT-PCR and western blot. HIF-1α follow-up damage.Our data that the expression levels of HIF-1α protein and miR-210 in brain tissue increased after LPS combined with HI injury( p 0.05), which is consistent with the HIF-1α( p <0.05). These results suggest that xenon can alleviate white matter damage by activating HIF-1α expression, and the stable expression of HIF-1α can regulate the expression of microRNA-210, which plays a neuroprotective role by inhibiting neuronal apoptosis through related pathways. The available time-window for treatment effectiveness and administration is an important aspect to consider in potential treatments for premature brain injury. Therefore, we investigated the effectiveness of xenon when administered immediately or delayed for 2 or 5 hours following LPS and HI-induced injury. The level of gene miR-210.The Xe therapeutic time window was within 5 hours after intervention, the sooner the

3 its target gene miR-210.The Xe therapeutic time window was within 5 hours after intervention, the sooner the better. Background WMD is a prominent neurological deficit observed in preterm birth. WMD is a leading cause of acute mortality and chronic morbidity. [1] Miller S P. previously reported that more than 25% of premature infants with brain injury may have different degrees of neurological sequelae,and the incidence of cerebral palsy is as high as 10%. [2]HI and inflammation are two main pathogenic mechanisms of brain injury in premature infants. [3,4] But there is no effective treatment for WMD at present. It is very important to explore a safe and effective intervention.
Xe is a noble gas used in general anesthesia. [5] Studies have shown that xenon has protective effects on ischemia-reperfusion injury of brain . [6,7] Moreover, xenon exhibited neuroprotective activity for the treatment of ischemic brain injury in combination with mild hypothermia in animal models. [8][9][10][11] Xenon is known to cross the blood-brain barrier (BBB) and has a low blood/gas solubility, which will reduce the risk of developing adverse effects. [12][13][14]Previous reports demonstrated that the anesthetic effect of xenon was primarily mediated through the inhibition of N-methyl-D-aspartate (NMDA) . White matter injury has become the main cause of nervous system dysplasia in premature infants, but there is no effective treatment for WMD at present. It is very important to explore a safe and effective intervention. [15,16] Particularly, Ma et al demonstrated that xenon could induce the activation of hypoxia-inducible HIF-1α and provide preconditioning effects on ischemic renal injury in mice. [17]However, the exact molecular mechanism of xenon-mediated neuroprotection remains to be elucidated. Moreover, the neuroprotective effect of xenon in WMD has not been adequately investigated.
MicroRNAs (miRNAs) are endogenous, non-coding small RNA molecules of about 22 nucleotides in length and are involved in regulating gene expression, primarily at the posttranscriptional level.
MiRNAs play a ubiquitous role in many vital biological processes, such as cell differentiation, proliferation, and apoptosis,etc. miR-210 is recognized as one of the most stable and significant microRNAs, and it is a stable target gene of HIF-1α,during hypoxia,HIF-1α is up-regulated,which 4 induces the expression of microRNA-210 to increase. [18]In this study, we investigated the neuroprotective effects of xenon in a neonatal rodent model of WMD. Further, we examined the impact of xenon on the outcome through measuring the expression of HIF-1α and miR-210 in neonatal brains.

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 humiditycontrolled 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.

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% O 2 and 92% N 2 ) 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.

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 6 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.

Protective effect of xenon following LPS and HI injury
First, we examined the efficiency of LPS and HI in inducing WMD in P3 rat pups as we have done previously [19]. In Group A ,HE staining of brain tissue demonstrated a normal white matter structure and morphology,regular cell arrangement with intact,centrally-positioned nuclei and a clear entoblast at 0, 24, 48 and 72 hours ( Fig.1 A-D, respectively).Upon the induction of WMD, HE staining revealed a distorted neural arrangement,loosened cortical structure, distorted nuclear membranes with pyknosis,as well as cellular degeneration and signs of necrosis at 48h in group B (Fig.1E-F).
Additionally,we observed an increase in the glial population along with tangled nerve fibers in group B at 72 hours ( Fig.1G-H).The abnormal structure and morphology observed in the white matter following LPS and HI indicate that LPS-sensitized HI-induced WMD in group B pups.Following the 3-hour xenon inhalation,the pathological changes induced by LPS and HI were less prominent in group C compared to group B supporting the notion that xenon could protect against brain injury in premature rats at 0,24,48 and 72 hours(Fig1 I-L, respectively)

Xenon alleviated the LPS and HI-induced apoptosis
Next,we performed TUNEL staining to estimate the apoptosis at 24, 48 and 72 hours following LPS and HI treatment.In the group A, TUNEL staining did not reveal significant apoptosis. In contrast, pups in group B showed a significant increase in apoptotic cells in the white matter at all examined time points( Fig.2 A-C). Further,the apoptosis rate was directly correlated with the survival period, i.e. the number of apoptotic cells significantly increased at 48h and peaked at 72 h following LPS and HI insult ( Fig.2 G). Following xenon treatment in group C, the number of apoptotic cells significantly decreased at different time points compared to group B which is Xenon untreated (P<0.05 at 24, 48 and 72 hours, respectively. Fig.2 D-F and G).

Xenon up-regulated the expression of miR-210 following white matter injury
To investigate the effect of xenon treatment on the WMD, we detected the miR-210 level by RT-PCR.
Compared with in group A, the expression level of miR-210 in neonatal rats' periventricular tissue increased significantly at all time points in group B (p<0.05). While the expression level of miR-210 in 8 brain tissues of group B was significantly lower at 48h and 72h than that of group C (p<0.05), but made no difference with that of group D or group E (p>0.05). Compared with group C, the expression of miR-210 in brain tissues of group D decreased significantly at 24h and 72h (p<0.05), and group E decreased significantly at 0h,24h,48h and 72h (p<0.05). And the expression of miR-210 at each group increased firstly and then decreased, and reached the peak at 48 hours. (table 1 and Fig.3)

Xenon up-regulated the expression of HIF-1α after white matter injury
Next, we investigated the expression of HIF-1α and the efficiency of xenon treatment. Compared to group A, Detection of HIF-1α protein by Western blot.The expression level of HIF-1α protein increased firstly and then decreased in each group, reaching peak at 24h, and was statistically differentiated with each other at every time point (p<0.05). The level of HIF-1α protein in group B brain tissues was significantly higher than that of group A at each time point,while significantly lower than that of group C and D and E at 0h, 24h and 72h (p<0.05). Compared with group C and group D, the expression of HIF-1α in the brain tissue of group E decreased significantly at 24h (p<0.05). (table 2 and Fig.4)

Discussion
The complex pathophysiology of WMD enables multiple targets at different time points of the diseaseprocess. For instance, in the early phase therapies are mainly concentrated on reduction of excitotoxic, oxidative and apoptotic mediators of injury. The development of therapies to reduce brain injury secondary to WMD is important because of the severity of disability that may result.
HIF-1α an important factor in the regulation of hypoxia, and has been proved to be involved in hypoxic-ischemic preconditioning in multiple species of tissues as a transcription factor.
Physiologically, HIF-1α is hydroxylated by prolyl-4-hydroxylases (PHDs) in the oxygen-dependent degradation domain of proline. After that,HIF-1α becomes ubiquitin ligase complex, which is degraded by proteasome. HIF-1α is degraded by this mechanism. Under normoxic conditions, HIF-1α protein has a very short half-life (less than 5 min under posthypoxic conditions in cell culture), and decreases in oxygen concentration cause its stability to increase almost immediately, as reduction of PHD activity leadind to degradation of proteasome. [22] HIF-1α targets a wide variety of genes,including genes involved in energy metabolism,angiogenesis,cell proliferation,and survival.among others, [23]and also 9 plays a role in hypoxic preconditioning in many organs by increasing the expression of HIF-1α , [24,25]especially in the brain, the role of HIF-1α is well recognized. Prass et al. [26] showed that hypoxia-induced HIF-1α DNA connectivity increased, leading to cerebral ischemic tolerance. Hypoxic stimulation up-regulates the expression of HIF-1α and EPO protein, produces reactive oxygen species, and plays an obvious neuroprotective role. [27] Recently, a number of microRNAs induced during hypoxia have been identified.One of these microRNAs, miR-210 is strongly induced by HIF-1α and has pleiotropic effects. [28] Fasanaro et al. reported that the expression level of miR-210 in endothelial cells was up-regulated in hypoxic environment, while promoting angiogenesis to a certain extent. [29] It is also closely related to hypoxic-ischemic diseases of the brain. Most studies have shown a direct link between the expression of miR-210 and hypoxia . [30][31][32][33][34] In particular,at the binding site of HIF-1α on its promoter,the expression of miR-210 was significantly up-regulated between normal and transformed cells.
[33] Some studies have shown that miR-210 plays an important role in cell survival during hypoxia as a highly up-regulated microRNA., [28,29]Under normoxic conditions, the presence of miR-210 is not effective, but when hypoxia occurs,the increase of miR-210 expression will reduce the effect of hypoxic environment on cell metabolism to a minimum. [35] miR-210 is a stable target of HIF-1α,that is activated under hypoxic environment, which induces the up-regulation of the expression of miR-210 . [17]Hypoxia can promote the expression of HIF-1α increasing at 4 hours, peaking at 8 hours and decreasing at 24 hours,and apoptosis increases significantly at 24 hours after hypoxia,accompanied by down-regulation of HIF-1α expression, that suggesting HIF-1α may play a protective role in regulating apoptosis. [36] Animal experiments and cell culture data showed that the level of miR-210 increased immediately after hypoxic injury, and then decreased for several days.
Along with ischemia and hypoxia, the level of HIF-1α also changed continuously. Therefore, the increase of miR-210 level after HI injury was regulated by HIF-1α and related to the time of injury. [28,37] Kelly [38]recently discovered the loop of miR-210 regulating HIF-1α and identified a new HIF-1α regulatory factor, glycerol-3-phosphate dehydrogenase 1-like (GPD1L). The induction of miR-210 by HIF-1α reduced the expression of GPD1L protein, thereby increasing the stability of HIF-1α. In conclusion, HIF-1α protein increased significantly in a certain period of time, peaked at 24 hours, and then gradually decreased to baseline level with the extension of time after HI injury in neonate rats.
The protective effect was enhanced and then weakened. If intervention treatment was given after HI,HIF-1α degradation was inhibited to protect brain tissue,and the expression of miR-210 is related to the level of HIF-1α protein, that will bring new ideas for the follow-up treatment of neonatal brain damage.Our data suggest that the expression levels of HIF-1α protein and miR-210 in brain tissue increased after LPS combined with HI injury(p 0.05), which is consistent with the above study.
Xenon is an NMDA receptor antagonist that has been precluded from the widespread clinical use as a general anesthetic due to its relatively high cost. [14,15,39] Xenon has well documented neuroprotective properties that were reported in models of premature brain injury, [7,10,11,39]as shown in our previous papers. [19]In this study, we investigated the acute neuroprotective outcomes of xenon in an in vivo model of premature brain injury. In accordance with previous reports, we opted to use a 3-hour xenon treatment interval. [21,40] The present results indicate that xenon has the greatest neuroprotective effect in the range of 37.5-50 vol%, which is consistent with previous studies that 50% xenon can provide sufficient neuroprotective effect . [41][42][43] On the contrary, others have shown that xenon at 75% volume concentration has the greatest neuroprotective effect and does not affect oxygenation [41]. However, in addition to neuroprotective effect, xenon at this concentration may have adverse side effects . [43] We choose a mixture of 50% xenon, 30% oxygen and 20% nitrogen to intervene,the mixture has 2.8× the density but almost the same viscosity as air . [44]In our study, following LPS and HI insult, we observed the expression of miR-210and HIF-1α elevated,and our results revealed that the administration of 50% xenon balanced with 30% oxygen and 20% nitrogen for 3 hours after injury resulted in a significant increase of miR-210 and HIF-1α(p<0.05).
These results suggest that xenon can alleviate white matter damage by activating HIF-1α expression, and the stable expression of HIF-1α can regulate the expression of microRNA-210, which plays a neuroprotective role by inhibiting neuronal apoptosis through related pathways. The available timewindow for treatment effectiveness and administration is an important aspect to consider in potential treatments for premature brain injury. Therefore, we investigated the effectiveness of xenon when administered immediately or delayed for 2 or 5 hours following LPS and HI-induced injury. The level of microRNA-210 did not increase significantly, but the level of HIF-1α protein did not decrease significantly.Maintaining and stabilizing the level of HIF-1α protein still served to protect brain tissue.
In addition, results revealed that the delay in xenon administration for 5 hours resulted in a significant increase of HIF-1α ( p>0.05), which may suggest that xenon still exerts its neuroprotective effects for up to 5 hours after injury,and the regulation between HIF-1α and microRNA-210 is related to the time of injury,and may suggest that xenon still exerts its neuroprotective effects for up to 5 hours after injury,the sooner the beter.
In summary, we showed that xenon is an efficient neuroprotective agent against premature brain

Availability of data and material
The data and analysis in this study could be reasonably acquired from the corresponding author.

Not applicable
Authors' contributions YXY, ZJX,JH had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept, design, and drafting of the article:YXY,ZJX,LXH Acquisition, analysis, and interpretation of data. Animal experiment: JJ,XHM, ZLL.
All authors have read and approved the manuscript.   The expression of HIF-1α in the brain tissue at different time points in groups A -E a Western blot analysis of HIF-1α expression at 0 h among the different groups, and bar chart representing the quantitative analysis of HIF-1α expression level normalized to GAPDH level, # p < 0.05 compared to group A, ▲ p < 0.05 compared to group B (n = 6). b Western blot analysis of HIF-1α expression among the different groups at 24 h ,and bar chart representing the quantitative analysis of HIF-1α expression level normalized to GAPDH level, # p < 0.05 compared to group A ▲ p < 0.05 compared to group B,* p < 0.05 compared to group C, △ p < 0.05 compared to group D (n = 6). c HIF-1α expression among the different groups at 48 h, and bar chart representing the quantitative analysis of HIF-1α expression level normalized to GAPDH level, # p < 0.05 compared to group A, ▲ p < 0.05 compared to group B (n = 6).d HIF-1α expression among the different groups at 72 h, and bar chart representing the quantitative analysis of HIF-1α expression level normalized to GAPDH level, # p < 0.05 compared to group A, ▲ p < 0.05 compared to group B (n = 6).

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