Xenon Upregulates Hypoxia Inducible Factor 1 Alpha in Neonatal Rat Brain under Normoxic Conditions

1 Department of Physiology and Biochemistry, School of Medicine, University of Pisa, Pisa, Italy 2 Department of Anesthesiology, University of Texas Medical School at Houston, 6431 Fannin Street, Houston, TX 77030, USA 3 Division of Surgical Molecular and Ultrastructural Pathology, School of Medicine, University of Pisa, Pisa, Italy 4 The Anaesthetics, Pain Medicine, and Intensive Care Section, Department of Surgery and Cancer, Imperial College London, Chelsea & Westminster Hospital, London, UK 5 Surgical Pathology Unit, Department of Oncology, ASL no. 1, Massa-Carrara, Italy


Introduction
Xenon has been approved in Europe as a medical gas, and its utility has been demonstrated through clinical and basic research studies.Although major interest focuses on xenon's hemodynamic and analgesic properties, increasing interest has recently focused on its potential long-lasting neuroprotective properties, which have been demonstrated both in in vivo and in vitro models of neuronal injury [1][2][3][4][5].Similar properties have been observed in the setting of anesthetic preconditioning [6][7][8], suggesting that xenon may play a useful clinical role in therapeutically unmet disease states such as stroke or neonatal asphyxia.Preconditioning is a biological process observed in several organs, in which a transient stimulus induces a protective state against a more prolonged, potentially lethal insult [9,10].Experimental studies into forms of anesthetic-induced preconditioning (APC), including hypoxia and sublethal ischemia, have indicated that preconditioning occurs in two waves (early and delayed) and with two potencies (short/immediate and long/deferred) [11].The early phase, mostly comprising phosphorylation steps, starts immediately after the application of the preconditioning stimulus and lasts for 2-3 hours [12], while the delayed phase is evident after 12-24 hours and lasts for up to 3 days.This long-lasting effect is the result of a transient alteration in gene transcription and depends on protein synthesis [13,14].Because it seems to be agent-, organ-, species-, and age-dependent [13,[15][16][17], further investigation into the mechanisms of preconditioning is warranted.
Studies of asphyxia and hypoxia-ischemia, and also isoflurane toxicity and apoptosis, suggest that xenon may be a valuable preconditioning and neuroprotective anesthetic agent that does not exert neurotoxic effects in the fragile developing brain [1,7,8,[16][17][18].Xenon induces early transcriptional changes in the brain of neonatal rodents exposed for 2 hours to the gas [19,20], and although an increase in mRNA does not always result in new protein synthesis, we previously demonstrated an increase in both signal and message of activity-dependent neuroprotective protein (ADNP), a key brain protein involved in several cell survival mechanisms, 24 hours after xenon exposure (the peak of delayed preconditioning) [19].
Hypoxia inducible factor 1 alpha (HIF-1α) is regulated by cellular oxygen tension [21] and is important in neuronal protection during hypoxia/ischemia and delayed preconditioning events.One of the signals upstream of HIF-1α is the mammalian target of rapamycin (mTOR), which regulates many activities including cell proliferation, cell survival, and protein synthesis; mTOR is inhibited by rapamycin [22].Although the effects of HIF-1α in xenon-mediated preconditioning have been confirmed extensively in an adult mouse model of kidney ischemia [23], the role of xenon-induced preconditioning in the developing brain remains unknown.
Given the importance of HIF-1α in mediating preconditioning events and the incomplete understanding of xenon's mechanism of action, our aim was to study the possible role of the mTOR-HIF-1α signalling axis in xenon-induced preconditioning.Using suppression subtractive hybridization (SSH), relative real-time polymerase chain reaction (PCR), and immunohistochemistry, the effects of xenon exposure on mTOR and HIF-1α transcript and protein levels were assessed during the peak of delayed preconditioning (24 hours postexposure).

Animal Handling.
This study was approved by the Animal Welfare Committee at the University of Pisa, and experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/ 609/EEC).All efforts were made to minimize animal suffering and the number of animals used.Twenty P7 Sprague-Dawley rats from two different litters were used.The two cohorts, xenon-exposed rats (experimental) and air-exposed rats (control), were randomly assigned (n = 10/cohort).The control group was exposed to 25% oxygen in 75% nitrogen (air group), and the treated group was exposed to 25% oxygen in 75% xenon.Xenon concentration was measured continuously by an in-line gas analyzer (Air Products model no.439Xe, Allentown, PA), while other gases were monitored through infrared gas analyzer (Datex Inc., Helsinki, Finland).The temperature of the exposure chambers was maintained at 37 • C via a water bath.After 2 hours of exposure, n = 6 rats from each cohort were immediately decapitated, and their forebrains were isolated in ribonuclease-free conditions, snap frozen in liquid nitrogen, and stored at −80 • C.

RNA Extraction.
Total ribonucleic acid (RNA) was isolated from the forebrains as previously described [20].Tissue samples were homogenized in an Ultra-Turrax homogenizer (IKA Works, Inc. Wilmington, NC) in the presence of a denaturing guanidium thiocyanate buffer.Homogenates were laid over a cesium chloride cushion and ultracentrifuged at 40,000 rpm for 24 hours (Beckman Ultracentrifuge L8-70 M; rotor Kontron TY65).The RNA pellet was dissolved in DEPC-treated water, precipitated overnight with 0.1 volume (v/v) of sodium acetate (3 M, pH of 6.4) and 2.2 volumes (v/v) of absolute ethanol, and solubilized in DEPC-treated water.The concentration and purity of isolated RNAs was determined spectrophotometrically (optical density 260/280 ratio between 1.8 and 2.0).For quality control purposes, total RNA was denatured with formaldehyde and size-fractionated through a 1% denaturing agarose gel, and visualized by ethidium bromide staining of the gel (28S/18S ratio between 1.5 and 2.5).The samples were then stored at −80 • C. Poly(A) + RNAs were isolated from the pools of total RNAs using the PolyATtract mRNA Isolation Kit (Promega, Madison, WI) according to manufacturer's protocol.Poly(A) + mRNA quality and integrity were assessed by RT-PCR (ImProm-II Reverse Transcription System, Promega, Madison, WI) using 3 specific primer sets for each recombinant deoxyribonucleic acid (DNA) gene (18S, 5.8S, and 28S, resp.) and one glyceraldehyde-3-phosphate dehydrogenase-(G3PDH-) specific primer set.

Suppression Subtractive
Hybridization.Suppression subtractive hybridization (SSH) was performed using the BD PCR-Select complementary DNA (cDNA) Subtraction Kit (BD Biosciences-Clontech, Palo Alto, CA) according to the manufacturer's protocol.The procedure required 0.5-1 μg of poly(A) + RNA from mRNA populations of both groups that was first converted to cDNAs.The cDNAs were used as tester (samples from xenon-exposed group) and driver (samples from control group) for the forward subtraction and vice versa for the reverse subtraction.Two cDNA libraries were thus obtained: the forward library containing genes positively modulated or switched on by xenon exposure and the reverse library containing genes negatively modulated or switched-off by xenon exposure.Tester and driver cDNAs were hybridized, and the hybrid sequences were removed.Consequently, the unhybridized cDNAs represented fragments corresponding to modulated or on/off switched sequences.The efficiency of subtraction was estimated by hybridization and PCR analyses.Hybridization analyses were carried out with Southern blot of cDNAs derived from subtracted and unsubtracted libraries.cDNAs derived from forward-and reverse-subtracted libraries were used as probes after being labelled with digoxigenin (DIG) DNA Labelling Kit (Roche, Mannheim, Germany).PCR analysis consisted of amplification of the G3PDH reference gene cDNA in subtracted and unsubtracted cDNAs, in order to evaluate its differential expression in subtracted and unsubtracted samples.Aliquots of the samples were drawn after 18, 23, 28, and 33 cycles of PCR amplification, and the products were then analyzed by agarose gel electrophoresis.Amplified cDNA sequences from the forward library were then directly inserted into a T/A-cloning vector using the TOPO TA Cloning Kit (Invitrogen, Milan, Italy).Because the final SSH products were enriched but not strictly composed of differentially expressed cDNAs, a differential screening was performed in order to sort out false positive clones.Positive clones were PCR amplified by colony PCR and then checked by 2% agarose gel electrophoresis. 1 μL of each heat-denatured PCR product was spotted onto positively charged nylon membrane (Roche, Mannheim, Germany) and UV crosslinked by Hoefer UVC 500 Ultraviolet Cross-linker (Amersham Biosciences, Piscataway, NJ).Two identical blots were created and hybridized with DIG-labelled forward and reverse library probes.In order to quantify the difference in signal intensity, spots were scanned using an image scanner, and signals were quantified with the Image Master 2D platinum software version 5.0 (Amersham Biosciences, Piscataway, NJ).Clones were subjected to differential screening, and those showing an on/off hybridization signal were sequenced by the dideoxy chain terminator method with an automated DNA sequencer (MGM, Institute of Molecular Genetics and Medicine, Pisa, Italy).Database searches were performed using the National Center of Biotechnology Information server (http://www.ncbi.nlm.nih.gov/) with BLAST algorithms at the European Bioinformatics Institute server (http://www.ebi.ac.uk/) and at the Rat Genome Database (http://rgd.mcw.edu/).

Data Validation and Expression
Analysis.SSH raw data validation was performed using relative RT-PCR on total RNA used for construction of libraries.PCR products were separated on 2% agarose gel electrophoresis, and band intensities were analyzed by Quantity One software version 4.4.1.(Bio-Rad, Hercules, CA).Validation of selected sequences and expression analysis were performed on the other (validation) animal cohort with relative real-time PCR (MGM, Institute of Molecular Genetics and Medicine, Pisa, Italy) using 7900HT FAST Real-Time PCR Sybr Green I Dyemethod (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions.Expression analysis of mTOR transcript was performed using semiquantitative RT-PCR on RNA from the validation set (Cyclophylin A used as reference gene).Cyclophylin A fragment amplification length was 351 bp, and mTOR fragment amplification length was 409 bp.Specific PCR primers were designed using Primer3 software (http://frodo.wi.mit.edu/primer3/) and subjected to a BLAST search to ensure that the sequences did not share high homology with identified mRNAs.100 ng of total RNA was used for reverse transcription using ImProm-II Reverse Transcription System (Promega, Madison, WI) and Oligo(dT) 15 primers according to the manufacturer's protocol.1 μL of 1 : 10 diluted reverse transcript was used as the PCR template.PCR products were separated with 2% agarose gel electrophoresis, and band intensities were analyzed by Quantity One software version 4.4.1.(Bio-Rad, Hercules, CA).

Immunohistochemistry.
Immunohistochemistry was performed on n = 4 rats/cohort to assess for relationships between gene transcription, protein translation, and localization at the cellular level.Twenty-two hours after the 2-hour xenon or air exposure, rats were euthanized with thiopental intraperitoneal injection (100 mg/kg body weight) and perfused with 4% paraformaldehyde.Brains were removed, postfixed overnight in perfusate, and processed for paraffin embedding.Serial coronal sections (6 μm thickness) were cut with a microtome, collected on super-frost slides, and dried overnight at 37 • C. Paraffin sections were stained with antibodies against HIF-1α (ab1, 1 : 100 dilution, Abcam, Cambridge, UK).Visualization of primary antibody was achieved with VECTASTAIN Elite ABC Kit (Vector Laboratories, Burlingame, CA) and diaminobenzidine tetrahydrochloride as substrate (K3466, DakoCytomation, Denmark), a technique based on the indirect streptavidin-biotin method.Slides were counterstained with hematoxylin.Staining intensity and morphologic qualitative assessment were carried out by two blinded independent observers using 20x and 40x magnification in two separate evaluation areas for each animal.Images were captured with the same exposure time for both groups at each magnification level.

Statistical Analysis. Statistical analysis of real-time PCR
data was performed using REST 2005 version 1.9.12 software (Corbett Research, Sydney, Australia).Analysis of relative mTOR/Cyclophilin A transcript from quantified real-time PCR gel images was performed using two-tailed Student's t-test (GraphPad Prism 4.00 software, GraphPad Software, La Jolla, CA).Statistical significance was defined as P < 0.05.Data are reported as mean ± standard deviation unless otherwise specified.

Results and Discussion
3.1.Results.Immunohistochemistry analysis of striatum sections from brains obtained 24 hours after the 2-hour exposure period revealed increased HIF-1α protein synthesis after xenon exposure (Figure 1).In sections from the control group, HIF-1α immunoreactivity was light in the neuronal cytoplasm and apparently absent in the neuronal nuclei.In sections from the xenon-exposed group, the presence of a strong and diffuse neuronal nuclear HIF-1α immunoreactivity was detected.Based on the morphological assessment of two independent experts (a neuropathologist and an anatomical histologist), this signal was clearly predominant in neurons and absent from glial cells.
During SSH, after the forward library was inserted into a T/A-cloning vector, 2208 white colonies were obtained.The differential screening of data obtained from SSH technique resulted in the selection of 43 positive clones.Their inserts were sequenced, and in silico analysis was performed using an online database as previously described [20].Based on its putative role in the cellular response to hypoxia and its suggested neuroprotective properties, the HIF-1α sequence was subjected to a more detailed analysis.There was a significant 2.3-fold upregulation in HIF-1α in the developing brains of xenon-exposed rats compared to air-breathing animals immediately after the 2-hour exposure period, P < 0.005 (Figure 2).In addition, the mTOR sequence was analyzed in detail due to its upstream relation to HIF-1α and its inhibition with rapamycin leading to loss of xenon-induced preconditioning through HIF-1α.A representative RT-PCR gel showing the amount of mRNA product of mTOR and Cyclophilin A in response to gas exposure is shown in Figure 3. Quantitative gene expression of RT-PCR analysis revealed a mTOR/ Cyclophylin A ratio of 0.67 ± 0.32 in the control group and 0.57 ± 0.36 in the xenon-exposed group (P < 0.44), indicating a lack of xenon effect on mTOR transcription after 2 hours of xenon exposure.

Discussion
. Hypoxic preconditioning refers to a brief period of hypoxia that induces tolerance to brain ischemia and that protects against lethal insults that occur minutes to days following hypoxia.This phenomenon has been related to the synthesis of new RNA and proteins, as well as the posttranslational modification of proteins, and several experimental studies suggest that ischemic tolerance induced by hypoxic preconditioning is due in part to hypoxic induction of HIF-1α and its target genes [24].The anesthetic gas xenon has been shown to have preconditioning effects (e.g., protecting cells from late ischemic and hypoxic events) in different preclinical and clinical settings [15,25].We focused our investigation on HIF-1α not only because it is the main hypoxia sensor in mammalian cells and activates oxygen level-dependent transcription, but also because it is the most important mediator of hypoxia preconditioning [24].Its transcriptional activity makes HIF-1α a major contributor to preconditioning in neonatal rat brain [26] and rat retinal neurons [27].We performed SSH to evaluate the effects of xenon-induced preconditioning in vivo in developing rat brain, under conditions that have previously resulted in preconditioning [16,20].A significant increase in HIF-1α mRNA transcript in response to xenon exposure was found in our previous study [20], and in the present study we confirm previous findings that xenon also upregulates HIF-1α protein synthesis [28], which is likely due to at least two signalling axes.The first axis involves the phosphoinositide 3-kinase (PI3K)-Akt-mTOR signalling pathway [28,29] while the second axis involves the mitogenactivated protein kinase (MAPK)-mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK)-extracellular signal-regulated protein kinase (ERK)-MAP kinaseinteracting serine threonine kinase (MNK) signalling pathway [8,15].Support for HIF-1α activation independent of mTOR or other messengers is increased because HIF-1α has an internal ribosomal entry site that allows for cap-independent translation [30].At the cellular membrane, xenon has been shown to require the opening of plasmalemmal adenosine triphosphate-sensitive potassium channels to induce neuronal preconditioning that have been shown to activate ERK [31], one of the downstream effectors of the MAPK signalling pathway.The protective effects of increased HIF-1α translocation demonstrated in neurons of the striatum are similar to the protective effects of xenon in developmental neuroapoptosis in rat and mouse brain [16,17].In mammalian cells, the expression of the α subunit of HIF-1α is maintained at low levels under normoxic conditions but is elevated during hypoxia, when HIF-1α undergoes protein stabilization and translocates from the cytoplasm into the nucleus, dimerizes with HIF-1β, and induces the transcription of a broad range of genes involved in increasing oxygen availability and facilitating acclimatization to low oxygen level conditions [32].Given that xenon exposure resulted in upregulation of HIF-1α under normoxic conditions, it is possible that xenon enables cells to withstand hypoxic conditions through signalling cascades that are not triggered by decreases in cellular oxygen tension.For example, HIF-1α has been shown to be regulated by other stimuli, including mechanical stress and growth factors [33].The effects of the 2.3-fold upregulation in HIF-1α upon exposure to xenon observed under normoxic conditions in this study need to be further studied and may include neuroprotection, as has been shown in the setting of hypoxia-ischemia [6] that results in endogenous HIF-1α upregulation.
Although this preliminary study provides novel results that differ from previously published works, it has a few important limitations.Firstly, the evaluation of other HIF-1α responsive genes in addition to mTOR (e.g., vascular endothelial growth factor) would have further supported a role for xenon exposure in upregulating HIF-1α.Furthermore, Western blot analysis of HIF-1α would have provided more sensitive information on posttranslational protein levels.Although the molecular effects of xenon on HIF-1α are modulated by the level of oxygenation, the investigation of different oxygen levels would have complicated the original scope of the present study.

Conclusions
In conclusion, xenon exposure during conditions of delayed preconditioning induced expression of HIF-1α mRNA, which was confirmed at the intracellular level, via molecular mechanisms possibly independent of mTOR signalling.Results of future studies after the current preclinical investigation may confirm the potential benefits of xenon as an anesthetic in the context of preconditioning and neuroprotection in brain ischemia.

Figure 1 :
Figure 1: Hypoxia inducible factor 1 alpha (HIF-1α) immunohistochemistry in striatum 24 hours following 2-hour exposure period.Immunohistochemistry on paraffin-embedded serial coronal sections (6 μm thickness), n = 2 sections/animal, n = 4 animals/group.For each magnification level, images were captured with the same exposure time for both groups; representative images are shown.(a) Control group brain (20x) reveals light immunoreactivity in neuronal cytoplasm and absent immunoreactivity in neuronal nuclei.(b) Xenon group brain (20x) reveals more immunoreactivity in neuronal cytoplasm compared to (a), and immunoreactivity in neuronal nuclei is present.(c) Control group brain (40x).(d) Xenon group brain (40x) reveals strong and diffuse nuclear immunoreactivity (arrowheads) with cytoplasmatic immunoreactivity.Scale bar represents 20 μm.