Neuroprotective Effect of Dexmedetomidine on Hyperoxia-Induced Toxicity in the Neonatal Rat Brain
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Research Article Neuroprotective Effect of Dexmedetomidine on Hyperoxia-Induced Toxicity in the Neonatal Rat Brain Marco Sifringer, 1 Clarissa von Haefen, 1 Maria Krain, 1 Nadine Paeschke, 1 Ivo Bendix, 2 Christoph Bührer, 3 Claudia D. Spies, 1 and Stefanie Endesfelder 3 1 Department of Anesthesiology and Intensive Care Medicine, Charit´ e-Universit¨ atsmedizin Berlin, Campus Virchow-Klinikum, 13353 Berlin, Germany 2 Department of Pediatrics I, Neonatology, University Hospital Essen, 45122 Essen, Germany 3 Department of Neonatology, Charit´ e-Universit¨ atsmedizin Berlin, 13353 Berlin, Germany Correspondence should be addressed to Marco Sifringer; [email protected] Received 7 October 2014; Accepted 10 December 2014 Academic Editor: Daniela Giustarini Copyright © 2015 Marco Sifringer et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Dexmedetomidine is a highly selective agonist of 2-receptors with sedative, anxiolytic, analgesic, and anesthetic properties. Neuroprotective effects of dexmedetomidine have been reported in various brain injury models. In the present study, we investigated the effects of dexmedetomidine on neurodegeneration, oxidative stress markers, and inflammation following the induction of hyperoxia in neonatal rats. Six-day-old Wistar rats received different concentrations of dexmedetomidine (1, 5, or 10 g/kg bodyweight) and were exposed to 80% oxygen for 24 h. Sex-matched littermates kept in room air and injected with normal saline or dexmedetomidine served as controls. Dexmedetomidine pretreatment significantly reduced hyperoxia-induced neurodegeneration in different brain regions of the neonatal rat. In addition, dexmedetomidine restored the reduced/oxidized glutathione ratio and attenuated the levels of malondialdehyde, a marker of lipid peroxidation, aſter exposure to high oxygen concentration. Moreover, administration of dexmedetomidine induced downregulation of IL-1 on mRNA and protein level in the developing rat brain. Dexmedetomidine provides protections against toxic oxygen induced neonatal brain injury which is likely associated with oxidative stress signaling and inflammatory cytokines. Our results suggest that dexmedetomidine may have a therapeutic potential since oxygen administration to neonates is sometimes inevitable. 1. Introduction Premature birth is the leading cause of child mortality and morbidity in preterm infants because most organs and also the antioxidant enzyme system are not fully developed struc- turally and functionally [1, 2]. Despite advances in perinatal medicine, whereby the chances of survival of premature infants could be significantly increased, in preterm infants with low birth weight impairment of brain development is oſten observed [3, 4]. A general anesthesia is sometimes essential for premature infants with medical indication. In pediatric anesthesia usu- ally the same anesthetics and adjuvants are used as in adults. NMDA antagonists like ketamine and/or GABA A receptor agonists, such as benzodiazepines, barbiturates, isoflurane, or propofol, are employed [5, 6]. In particular, premature and newborn infants demonstrate a significantly higher risk of anesthesia [7] and perioperative morbidity and mortality is increased due to the immature organ systems [8]. In addition to required medical interventions under sedation also high oxygen concentrations are a major problem. In recent years, experimental studies and clinical observations showed that oxygen, which is widely used in neonatal intensive care for treatment of respiratory distress, triggers a disruption of intracellular redox homeostasis and disturbed neurological development of preterm infants [9–11]. As we demonstrated recently in experimental models, this disturbance can induce oxidative stress by modulation of the glutathione ratio and an increasing lipid peroxidation [12–15], inflammation by increased levels of proinflammatory cytokines [13, 15, 16], leading to increased neurodegeneration [12, 16, 17], and Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity Volume 2015, Article ID 530371, 10 pages http://dx.doi.org/10.1155/2015/530371
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Transcript of Neuroprotective Effect of Dexmedetomidine on Hyperoxia-Induced Toxicity in the Neonatal Rat Brain
Research ArticleNeuroprotective Effect of Dexmedetomidine onHyperoxia-Induced Toxicity in the Neonatal Rat Brain
Marco Sifringer1 Clarissa von Haefen1 Maria Krain1 Nadine Paeschke1 Ivo Bendix2
Christoph Buumlhrer3 Claudia D Spies1 and Stefanie Endesfelder3
1Department of Anesthesiology and Intensive Care Medicine Charite-Universitatsmedizin Berlin Campus Virchow-Klinikum13353 Berlin Germany2Department of Pediatrics I Neonatology University Hospital Essen 45122 Essen Germany3Department of Neonatology Charite-Universitatsmedizin Berlin 13353 Berlin Germany
Correspondence should be addressed to Marco Sifringer marcosifringercharitede
Received 7 October 2014 Accepted 10 December 2014
Academic Editor Daniela Giustarini
Copyright copy 2015 Marco Sifringer et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Dexmedetomidine is a highly selective agonist of 1205722-receptors with sedative anxiolytic analgesic and anesthetic propertiesNeuroprotective effects of dexmedetomidine have been reported in various brain injurymodels In the present studywe investigatedthe effects of dexmedetomidine on neurodegeneration oxidative stress markers and inflammation following the induction ofhyperoxia in neonatal rats Six-day-old Wistar rats received different concentrations of dexmedetomidine (1 5 or 10120583gkgbodyweight) and were exposed to 80 oxygen for 24 h Sex-matched littermates kept in room air and injected with normal saline ordexmedetomidine served as controls Dexmedetomidine pretreatment significantly reduced hyperoxia-induced neurodegenerationin different brain regions of the neonatal rat In addition dexmedetomidine restored the reducedoxidized glutathione ratio andattenuated the levels of malondialdehyde a marker of lipid peroxidation after exposure to high oxygen concentration Moreoveradministration of dexmedetomidine induced downregulation of IL-1120573 on mRNA and protein level in the developing rat brainDexmedetomidine provides protections against toxic oxygen induced neonatal brain injury which is likely associated with oxidativestress signaling and inflammatory cytokines Our results suggest that dexmedetomidine may have a therapeutic potential sinceoxygen administration to neonates is sometimes inevitable
1 Introduction
Premature birth is the leading cause of child mortality andmorbidity in preterm infants because most organs and alsothe antioxidant enzyme system are not fully developed struc-turally and functionally [1 2] Despite advances in perinatalmedicine whereby the chances of survival of prematureinfants could be significantly increased in preterm infantswith low birth weight impairment of brain development isoften observed [3 4]
A general anesthesia is sometimes essential for prematureinfants with medical indication In pediatric anesthesia usu-ally the same anesthetics and adjuvants are used as in adultsNMDA antagonists like ketamine andor GABAA receptoragonists such as benzodiazepines barbiturates isoflurane orpropofol are employed [5 6] In particular premature and
newborn infants demonstrate a significantly higher risk ofanesthesia [7] and perioperative morbidity and mortality isincreased due to the immature organ systems [8] In additionto required medical interventions under sedation also highoxygen concentrations are a major problem In recent yearsexperimental studies and clinical observations showed thatoxygen which is widely used in neonatal intensive care fortreatment of respiratory distress triggers a disruption ofintracellular redox homeostasis and disturbed neurologicaldevelopment of preterm infants [9ndash11] As we demonstratedrecently in experimental models this disturbance can induceoxidative stress by modulation of the glutathione ratio andan increasing lipid peroxidation [12ndash15] inflammation byincreased levels of proinflammatory cytokines [13 15 16]leading to increased neurodegeneration [12 16 17] and
Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2015 Article ID 530371 10 pageshttpdxdoiorg1011552015530371
2 Oxidative Medicine and Cellular Longevity
inhibition of neuronal maturation [17 18] in the developingbrain
Dexmedetomidine is a potent and highly selective agonistof 1205722-receptors with sedative anxiolytic analgesic and anes-thetic properties [19ndash21] In addition it is generally reportedthat dexmedetomidine has neuroprotective effects in differ-ent animal models [22ndash27] and minimal side effects on therespiratory tract and the gastrointestinal function that min-imizes the exposure to other narcotics and benzodiazepines[23 28ndash30] Recent studies suggest the 1205722-adrenoceptor ago-nist dexmedetomidine to attenuate anesthetic agent inducedneuroapoptosis [26 31 32] and it appears for long termsedation as an alternative tomidazolam and propofol [33 34]Advantages of dexmedetomidine are shorter ventilation andrecovery times and lower hypertension and tachycardia Inparticular the shortening of the duration of ventilation seemsto be relevant in connection with the higher oxygen toxicityby prolonged ventilation and the associated development ofchronic lung disease and the consequent motor and cognitivedeficits in preterm born infants First results of clinical trialsin preterm infants revealed a decrease in the duration ofmechanical ventilation by half and no need for additionalsedative administration [35 36]
The aim of this study was to investigate the effect of dif-ferent concentrations of dexmedetomidine to the immaturebrain in a neonatal rat model of oxygen toxicity on neurode-generation oxidative stressmarkers and the expression of theproinflammatory cytokine IL-1120573
2 Materials and Methods
21 Animals and Drug Administration All procedures wereapproved by the State Animal Welfare Authorities (LAGeSoG014513) and followed institutional guidelines Six-day-old Wistar rats from time-pregnant mothers were obtainedfrom Charite-Universitatsmedizin Berlin (Germany) andrandomly assigned to cages and treatment
Dexmedetomidine (DEX dexdor Orion Pharma EspooFinland) was dissolved in phosphate buffered saline Threedoses of the drug (1 5 and 10 120583gkg body weight) wereused and all injections were given intraperitoneally (ip)as a fixed proportion of body weight (100 120583L10 g) The ratpups are divided into different biological groups (descriptionwith the relevant experimental abbreviations) (1) controlgroup (CON 21 O
2 room air) with 09 saline (2) verum
group (21 O2) with 1 120583gkg DEX (DEX1) (3) verum group
with 5 120583gkg DEX (DEX5) (4) verum group with 10 120583gkgDEX (DEX10) (5) hyperoxia group (HY 80 O
2 OxyCycler
BioSpherix Lacona NY) with 09 saline (6) hyperoxiawith 1 120583gkg DEX (HYDEX1) (7) hyperoxia with 5120583gkgDEX (HYDEX5) and (8) hyperoxia with 10 120583gkg DEX(HYDEX10) each with a number of six animals per groupand different gender For hyperoxia or normoxia exposurepups were kept together with their dams Saline or DEXwas administrated once 15min before the start of oxygenexposure
22 Tissue Preparation At 24 h of exposure the animals wereanaesthetized with an ip injection of ketamine (50mgkg)
xylazine (10mgkg) and acepromazine (2mgkg) 5minbefore being perfused For molecular analysis pups weretranscardially perfused with normal saline (pH 74) and thendecapitated the olfactory bulb and cerebellumwere removedand brain hemispheres were snap-frozen in liquid nitrogenand stored at minus80∘C For immunohistochemical analysisanimals were perfused with PBS followed by perfusion with4 paraformaldehyde at pH 74 and the brains were postfixedat 4∘C for 3 days embedded in paraffin and processed forhistological staining
23 DNA Fragmentation Assay Paraffin-embedded sectionswere cut (5 120583m) deparaffinized in Roti-Histol (Carl RothKarlsruhe Germany) twice for 10min each rehydrated indescending ethanol series and rinsed in phosphate bufferedsaline for 3min each at room temperature After deparaf-finization of sections an in situ detection of cells withDNA-strand breaks was performed by the TUNEL labelingmethod using a TdT-FragEL DNA fragmentation detectionkit (Millipore Darmstadt Germany) according to the manu-facturerrsquos instructions Negative controls were performed bysubstituting Tris-buffered saline for the TdT enzyme
The TUNEL positive cells were analyzed in frontal cor-tex (FC) retrosplenial cortex (RSC) hypothalamus (HTH)thalamus (TH) and the hippocampus Sections were viewedby light microscopy while blinded using a LEICA DM 2000microscope equipped with a 200x magnification TUNELpositive cells were counted in the anatomical regions of thebrain in up to twelve different sections per animal and region
24 Determination of Total Glutathione (GSH and GSSG)Total glutathione (GSH and GSSG) was measured in brainhomogenates using the thiol reagent 551015840-dithiobis-2-nitrob-enzoic acid (DTNB) as shown previously [14] In brief for thedetermination of reduced glutathione (GSH) and oxidizedglutathione (GSSG) the brains were homogenized and thehomogenates were treated with a mixture of metaphosphoricacid EDTA and NaCl After centrifugation aliquots weretaken for neutralization with disodium hydrogen phosphatefollowed by addition of DTNB GSH was determined afterreaction with DTNB in a spectrophotometer at 412 nmFor the determination of GSSG 4-vinylpyridine was addedand then incubated for 1 hour at room temperature 4-Vinylpyridine is able to mask the GSH content withoutinterfering with the spectrophotometrical determination ofGSSG at 412 nm GSH and GSSG levels are reported asnmolmg protein
25 Measurement of Lipid Peroxidation Lipid peroxidationwas determined by the reaction of thiobarbituric acid withmalondialdehyde (MDA) a product of lipid breakdowncaused by oxidative stress as previously described [14]A SUPELCOSIL LC-18-DB HPLC reversed-phase column(Sigma-Aldrich Munich Germany 5120583m particle size 250times 10mm ID) was utilized for the detection of MDA levelsin brain homogenates The MDA level was determined byfluorescence (525550 nm) with a 50mM potassium phos-phate buffer (pH 68) and 40 methanol mobile phase at15mLmin flow rate
Oxidative Medicine and Cellular Longevity 3
26 RNA Extraction and Semiquantitative Real-Time PCRTotal RNA was isolated from snap-frozen tissue by acidicphenolchloroform extraction (peqGOLD RNAPurePEQLAB Biotechnologie Erlangen Germany) and 2 120583g ofRNA was reverse-transcribed The PCR products of IL-1120573and hypoxanthine-guanine phosphoribosyltransferase (HPRTas internal standard) were quantified in real time usingdye-labeled fluorogenic reporter oligonucleotide probesand primers (Metabion Munich Germany) with the fol-lowing sequences and corresponding GenBank accessionnumbers IL-1120573 (NM 031512) sense 51015840-AACAAAAAT-GCCTCGTGCTGTCT-31015840 antisense 51015840-TGTTGGCTTATG-TTGTGTCCATTG-31015840 probe 51015840-ACCCATGTGAGCTGA-AAGCTCTCC-31015840 HPRT (NM 012583) sense 51015840-GGAAAG-AACGTCTTGATTGTTGAA-31015840 antisense 51015840-CCAACA-CCTTGAGAGGTCCTTTT-31015840 and probe 51015840-CTTTCC-CTTGGTCAAGCAGTACAGCCCC-31015840 All probes werelabeled at their 51015840 ends with the reporter dye 6-carboxy-fluoresceine (FAM) and at their 31015840 ends with the quencherdye 6-carboxy-tetramethylrhodamine (TAMRA) Real-timePCR and detection were performed in triplicate and repeated3 times for each sample using a total reactive volume of13 120583L which contained 65120583L of 2x TaqMan Universal PCRMaster Mix (Applied Biosystems Foster City CA USA)25 120583L of 125 120583M oligonucleotide mix 05 120583L (05 120583M) ofprobe and 50 ng of cDNA template The PCR amplificationwas performed in 96-well optical reaction plates for 40cycles with each cycle at 94∘C for 15 s and 60∘C for 1minThe expression of IL-1120573 and HPRT was analyzed with thereal-time PCR ABI Prism 7500 sequence detection system(Applied Biosystems) according to the 2minusΔΔCT method [37]
27 Immunoblotting Snap-frozen brain tissue was homog-enized in RIPA buffer solution for protein extraction Thehomogenate was centrifuged at 1050 g (4∘C) for 10min andthe microsomal fraction was subsequently centrifuged at17000 g (4∘C) for 20min After collecting the supernatantprotein concentrations were determined using the PierceBCA kit (Pierce Rockford IL USA) with a 30min incuba-tion at 37∘C prior to spectrophotometry at 562 nm Proteinextracts (30 120583g per sample) were denatured in Laemmlisample loading buffer at 95∘C separated by 15 of sodiumdodecyl sulfate polyacrylamide gel electrophoresis and elec-trotransferred in transfer buffer to a nitrocellulosemembrane(02 120583m pore Protran Schleicher amp Schull Dassel Ger-many) Nonspecific protein binding was prevented by treat-ing the membrane with 5 nonfat dry milk in Tris-bufferedsaline01 Tween 20 for 1 h at room temperature Equalloading and transfer of proteinswas confirmedby staining themembranes with Ponceau S solution (Fluka Buchs Switzer-land) The membranes were incubated overnight at 4∘C withrabbit polyclonal anti-IL-1120573 (17 kDa 1 1000 PromoKineHeidelberg Germany) Horseradish peroxidase-conjugatedsecondary anti-rabbit antibody was diluted 1 2000 (Amer-sham Biosciences Bucks United Kingdom) Positive signalswere visualized using the SuperSignal West Pico kit (Pierce)according to the manufacturerrsquos directions and quantifiedusing a ChemiDoc XRS+ system and the software Image Lab(Bio-RadMunich Germany)Membranes were stripped and
then washed blocked and reprobed overnight at 4∘C withmouse anti-120573-actin monoclonal antibody (42 kDa 1 10000Sigma-Aldrich) Each experiment was repeated three times
28 Statistical Analyses All data are expressed as mean plusmnstandard error of the mean (SEM) Groups were comparedusing a one-way analysis of variance (ANOVA) and sig-nificance was determined using Bonferronirsquos correction formultiple comparisonswith independent sample 119905-test A two-sided 119875 value lt 005 was considered to be significant Allgraphics and statistical analyses were performed using theGraphPad Prism 60 software (GraphPad Software La JollaCA USA)
3 Results
31 Dexmedetomidine Ameliorates Hyperoxia-Induced Neu-rodegeneration in the Infant Brain That oxidative stress isa trigger of cell death is well known We have investigatedthe changes in oxidative stress on the apoptotic cell deathby TUNEL assay These investigations were carried out inthe frontal cortex (FC) and retrosplenial cortex (RSC) inthe deep graymatter (hypothalamus (HTH) thalamus (TH))and in the hippocampus
In detail exposure to 24 h of hyperoxia (HY) from P6 toP7 resulted in a large increase of TUNEL positive cells andthe antiapoptotic ability of dexmedetomidine was able to bedetected by a significant decrease in the investigated differentconcentrations (FC HY 5029 + 130 versus HYDEX12307 + 195 HYDEX5 1550 + 272 and HYDEX10 621+ 72 RSC HY 4483 + 253 versus HYDEX1 1968 +312 HYDEX5 2034 + 246 andHYDEX10 1422 + 259HTH HY 5029 + 450 versus HYDEX1 1426 + 169HYDEX5 1699 + 325 and HYDEX10 1387 + 162 THHY 4424 + 423 versus HYDEX1 1955 + 332 HYDEX52156 + 187 and HYDEX10 1975 + 419) compared tocontrol animals (CON Figures 1(a) and 1(b)) ParticularlyDEX10 under hyperoxic conditions can reduce apoptotic cellrate in the cortex and hypothalamus to control level and sig-nificantly among them in the frontal cortex In the thalamusa significant reduction by DEX under oxygen exposure wasdemonstrated but did not reach the controls DEX undernormoxic conditions showed a significant increase of TUNELpositive cells in the cortices with DEX10 and in the thalamuswith DEX all over (Figure 1(b)) The results of the dentategyrus show a similar antiapoptotic effect of DEX (data notshown) A statistical evaluation of the dentate gyrus was notpossible due to low cell counts
32 Treatment with Dexmedetomidine Modifies Hyperoxia-Affected Levels of Reduced (GSH) and Oxidized Glutathione(GSSG) in the Developing Brain As we showed previ-ously neonatal oxygen toxicity causes an imbalance of theglutathione-redox-system correlating with an increase ofneurodegeneration in the immature brain [12ndash15]
Analysis of GSH and GSSG levels by reaction withthe classical thiol reagent DTNB and spectrophotometricalmeasurement at 412 nmwas performed in samples from totalbrain extracts of 7-day-old rats (119899 = 6 per group) exposed
4 Oxidative Medicine and Cellular Longevity
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Oxidative Medicine and Cellular Longevity 5
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Figure 1 Apoptosis caused by hyperoxia is prevented by dexmedetomidine (a) Representative TUNEL staining images (originalmagnification times400) of rat brain frontal cortices (FC) retrosplenial cortices (RSC) hypothalamus (HTH) and thalamus (TH) of P7 controlpups in room air without (CON) and with dexmedetomidine administration (DEX1 DEX5 and DEX10 corresponding to the concentrationsof 1 5 and 10120583gkg) and after 24 h of hyperoxia from P6 to P7 without (HY) andwith dexmedetomidine administration (HYDEX1 HYDEX5and HYDEX10) (b) Quantitation of TUNEL positive cells in the rat brain frontal cortices (FC) retrosplenial cortices (RSC) hypothalamus(HTH) and thalamus (TH) showed that relative to the control (white bars) hyperoxia at 24 h significantly increased these cell counts in cortexand deep grey matter (black bars) These levels were significantly decreased through systemic dexmedetomidine pretreatment (hatched greybars DEX 1 5 and 10120583gkg) However dexmedetomidine administration resulted in increased TUNEL positive cells in control rats mostprofound in TH (grey bars DEX 1 5 and 10120583gkg) Data are expressed relative to the normoxia-exposed control group (white bars 100)Bars represent mean + SEM 119899 = 6 per group lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus normoxiacontrol 119875 lt 001 and
119875 lt 0001
versus hyperoxia
to (i) normoxia and saline injections (CON) (ii) hyperoxiaand saline injections (HY) (iii) hyperoxia andDEX (HYDEX1 5 or 10 120583gkg) and (iv) normoxia and DEX (DEX 15 or 10 120583gkg) 24 h of hyperoxia triggered the decreaseof GSH levels in brain extracts (Figure 2(a) CON 1142 +011 nmolmg protein and HY 979 + 006 nmolmg protein)When DEX was administered together with hyperoxia itsignificantly increased the levels of GSH at a concentration of5 and 10 120583gkg DEX (HYDEX5 1019 + 006 nmolmg proteinand HYDEX10 1116 + 009 nmolmg protein)
Figure 2(b) shows the increased level of GSSG in thedeveloping brain after 24 h of hyperoxia (CON 0273 +0003 nmolmg protein and HY 0046 + 0007 nmolmgprotein) DEX coapplication reduced the hyperoxia-inducedincrease of GSSG levels significantly at 5 and 10 120583gkg DEX(HYDEX5 0398 + 0008 nmolmg protein and HYDEX100255 + 0008 nmolmg protein)
In total hyperoxia triggered the decrease of the GSHGSSG ratio in the developing rat brain (Figure 2(c) CON4194 + 078 and HY 2152 + 040) When DEX was adminis-tered at the start of hyperoxia the levels of GSHGSSG ratiosignificantly increased at 5 and 10120583gkg of DEX (HYDEX52563 + 060 and HYDEX10 4395 + 162)
33 Induction of Lipid Peroxidation by Hyperoxia in theNeonatal Brain Is Attenuated by Dexmedetomidine TreatmentTo demonstrate that the changes obtained in the level of GSHand GSSG may be connected with an altered lipid perox-idation we examined immature rat brains after treatmentof hyperoxia normoxia dexmedetomidine andor normalsaline treatment for the concentration of malondialdehyde(MDA) Increased MDA levels as a sign of lipid breakdownwere evident in rat brains at 24 h of hyperoxiawhen comparedto normoxic animals whereas a single DEX administrationbefore beginning hyperoxia exposure reduced these levelssignificantly at 5 and 10 120583gkg DEX (Figure 3 CON 563 +014 nmolmg protein and HY 1457 + 016 nmolmg proteinHYDEX5 1362 + 019 nmolmg protein and HYDEX101018 + 031 nmolmg protein)
34 Dexmedetomidine Treatment Reduces IL-1120573 ExpressionunderHyperoxic Conditions in the Infant Brain Aspreviouslyshown by our group hyperoxic conditions lead to an increasein IL-1120573 expression in the immature brain [13 15 16]
Hyperoxia triggered an increase of IL-1120573mRNA in brainhomogenates of neonatal rats as shown by semiquantitativereal-time PCR (Figure 4(a) HY 46599 + 6469) Control
6 Oxidative Medicine and Cellular Longevity
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Figure 2 Effect of dexmedetomidine on hyperoxia-modified GSH and GSSG levels in the developing brain (a) Reduced GSH levels wereevident in total rat brain extracts 24 h after the initiation of hyperoxia (black bar) when compared to normoxic animals (white bar) Theselevels were increased through dexmedetomidine (DEX) pretreatment in a concentration dependent manner (hatched grey bars 1 5 and10120583gkg) (b) Increased levels of oxidized GSSG were obvious in total brain extracts at 24 h of hyperoxia (black bar) when compared withnormoxic control animals (white bar)These levels were decreased through pretreatment with DEX (hatched grey bars 1 5 and 10120583gkg) (c)ReducedGSHGSSG ratio levelswere evident in rat brain extracts at 24 h of hyperoxia (black bar)when compared to normoxic control animals(white bar)These levels were upregulated throughDEX pretreatment (hatched grey bars 1 5 and 10120583gkg) Application of dexmedetomidineunder room air (grey bars) showed no effect on GSH or GSSG levels Bars represent mean + SEM 119899 = 6 per group lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 0001 versus hyperoxia
Oxidative Medicine and Cellular Longevity 7
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Figure 3 Alteration of lipid peroxidation by hyperoxia in the imma-ture brain Hyperoxia lead to a significant increase of MDA levelsafter 24 h of oxygen exposure (black bar) whereas a single DEXapplication of 5 or 10 120583gkg (hatched grey bars) before hyperoxiaexposure reduced these levels significantly Bars represent mean +SEM 119899 = 6 per group lowast119875 lt 005 and lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 001 and
119875 lt 0001 versus hyperoxia
animals showed low mRNA levels of IL-1120573 (CON 10036 +1141) When dexmedetomidine was administered togetherwith hyperoxia it significantly ameliorated the expression ofproinflammatory IL-1120573 depending on theDEX concentration(HYDEX5 31029 + 2177 and HYDEX10 17766 + 2233)on mRNA levels in rat pups Pretreatment with DEX inanimals kept under normoxic conditions had no effect onIL-1120573 gene expression (DEX1 11718 + 1158 DEX5 12141 +914 and DEX10 13531 + 1285)
Western blotting demonstrated that protein expression ofIL-1120573 is significantly increased following oxygen treatmentfor 24 h (Figure 4(b) HY 33666 + 1725) compared tocontrol animals (CON 10000 + 731) A significant effect onIL-1120573 protein levels was seen upon a single dexmedetomidineapplication of 5 or 10 120583gkg DEX in hyperoxia-exposedanimals (HYDEX5 25096 + 1355 and HYDEX10 17105+ 1227) Animals under normoxic conditions and DEXpretreatment showed low protein levels of IL-1120573 (DEX1 11195+ 443 DEX5 10786 + 515 and DEX10 11244 + 543)
4 Discussion
In the present study we demonstrate that the 1205722-adren-oceptor agonist dexmedetomidine leads to a decrease ofneurodegeneration and affects the level of oxidative stressparameters and the proinflammatory cytokine IL-1120573 in amodel of neonatal hyperoxia-induced brain injury
As shown previously oxygen toxicity leads to an increasedneurodegeneration in the immature brain [12 16 17 38] Inline with these studies here we indicate that an exposure ofsix-day-old rats to a high oxygen concentration (FiO
280)
over 24 h resulted in an increase of neurodegeneration to theneonatal brain (Figures 1(a) and 1(b))Moreover we point outa significant decrease of cell death in the developing rat brainwhen infant rats were treated intraperitoneally with 1 5 or10 120583gkg dexmedetomidine before exposure to high oxygenconditions These dosages are atunder the lower levels ofdexmedetomidine concentrations used in common adult ratmodels of neuronal damage [39 40] and the present dataare not intended to demonstrate the evidence of an effectivedose However our findings conform with different otheranimal studies which examined the neuroprotective capac-ity of dexmedetomidine [22ndash27] Hyperoxia-exposed ratsrevealed an up to 5-fold increase in cell degeneration withinthe cortex and deep gray matter (Figure 1(b)) which hasbeen demonstrated in previous studies [17 41] Interestinglydexmedetomidine treatment showed a profound decrease ofdegenerating cells in all analysed brain regions of rats exposedto hyperoxia (Figure 1(b)) Dexmedetomidine alone inducedno oxidative stress but at a relatively low concentration ofdexmedetomidine under normoxic conditions a significantincrease in apoptosis in the cortex and the thalamus hasbeen shown This result is controversial to other inventions[31 32 42] and to the description of only protective andno toxic effects [24 25 43] In vitro it was shown byKuhmonen et al [22] that dexmedetomidine is more effectiveat lower doses and protects against delayed cell death In anischemia-reperfusionmodel [39] antiapoptotic proteins wereupregulated and proapoptotic proteins are suppressed bydexmedetomidine treatment In the study by Li et al there areno changes in the expression of proapoptotic mediators in anisoflurane-induced model of neuroapoptosis [40] A provenhigh blood pressure after the administration of high-dosedexmedetomidine correlates with cerebral hypoperfusionmay be due to alpha-2-induced cerebral vasoconstriction[44] so that this could contribute to apoptotic cell deathtriggered by other cellular mechanisms However in ourstudy dexmedetomidine also led to negative effects in controlanimals notably in the thalamus and potential side effectsshould be further considered in studies on protection of theimmature brain using dexmedetomidine
The potency of dexmedetomidine in this model of oxy-gen toxicity indicates that the 1205722-adrenoceptor agonist hasantioxidant activities Of note our hyperoxia model is pre-dominantly based on free radical generation as we previouslyfound an induction of different oxidative stress parameters[12ndash15] Therefore we evaluated the effects on biochemicalmarkers of oxidative stress in the developing brain Theconcentrations of GSH and GSSG the GSHGSSG ratio andthe MDA level were used as indicators of oxidative stressand lipid breakdown Hyperoxia-exposed P6 rats showedsignificant decreased GSH and increased GSSG levels at24 h of hyperoxia In addition a decrease in GSHGSSGratio and an increase in MDA level in the developingbrain indicate neuronal damage due to oxidative stressbut there was a modulation of the levels of all parameters
8 Oxidative Medicine and Cellular Longevity
0
100
200
300
400
500
600
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowast
IL-1120573 gene expression
Rela
tive I
L-1120573
gene
expr
essio
n2minusΔΔ
Ct(
)
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(a)
0
100
200
300
400
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
IL-1120573 protein expression
Den
sity
ratio
IL-1120573
120573-a
ctin
( o
f con
trol)
IL-1120573 17kDa
120573-actin 42kDa
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(b)
Figure 4 (a) Quantitative analysis of mRNA expression by real-time PCR showed a marked increase of IL-1120573 mRNA expression in thebrain of P6 rat pups that were kept for 24 h under hyperoxia (black bar) whereas dexmedetomidine treatment restores IL-1120573 upon controllevel (hatched grey bars) depending on the dexmedetomidine concentration Application of dexmedetomidine under room air (grey bars)showed no significant regulation on IL-1120573 mRNA expression (b) The analysis of IL-1120573 protein expression by western blot showed a similarexpression pattern The protein expression of IL-1120573 is significantly increased after 24 h of hyperoxia and a single application of 5 or 10 120583gkgdexmedetomidine could restore the IL-1120573 protein expression almost up to control level The densitometric data represent the ratio of thepixel intensity of the IL-1120573 band to the corresponding 120573-actin band Blots are representative of a series of three blots Data are normalizedto levels of rat pups exposed to normoxia (CON = 100 white bars) Bars represent mean + SEM 119899 = 6 per group lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 005 119875 lt 001 and
119875 lt 0001 versus hyperoxia
up to normoxia levels in hyperoxia-exposed neonatal ratsthat received dexmedetomidine (Figures 2 and 3) Theseobservations suggest that dexmedetomidine seemingly hasan antioxidant activity against hyperoxia-induced oxidativestress in the developing brain In line with our findings thereare several studies that suggest an increased antioxidativecapacity of dexmedetomidine in the brain [24] and furtherorgans (reviewed by Tse et al [45])
This study indicates that dexmedetomidine significantlydecreases hyperoxia-induced IL-1120573 upregulation in the devel-oping rat brainThese results illustrate the protective effect ofdexmedetomidine on neuroinflammation as shown before inclinical and experimental studies [46 47] Dexmedetomidineitself did not affect IL-1120573 levels indicating that the 1205722-adrenoceptor agonist only acts to normalize the inducedproinflammatory cytokine (Figure 4) Here dexmedetomi-dine appears to inhibit the production of inflammatorymediators from multiple cell types for example astrocytesand microglia [48 49]
There are several limitations of this study pointing to areasof future investigations Dexmedetomidine was given onlyonce to the animals while preterm infants receive an initialloading dose and subsequent infusions [50] and the highdoses of dexmedetomidine studied here exceed those dosesrecommended for clinical use short term hyperoxia wasexamined and preterm infants are often exposed to a longerperiod of supraphysiological oxygen concentrations [51] andwe have not determined the plasma levels of dexmedeto-midine Further details of dexmedetomidine-induced sideeffects remain to be elucidated
To our knowledge this is the first report demonstratingthat dexmedetomidine mediates a decrease of neurodegen-eration and oxidative stress and affects the expression of theproinflammatory cytokine IL-1120573 in the hyperoxia-exposedneonatal brain Since oxidative stress and inflammation areinvolved in dysfunction of the immature brain under highoxygen conditions 1205722-adrenoceptor activation seems to bea potential neuroprotective treatment
Oxidative Medicine and Cellular Longevity 9
5 Conclusion
The essential core of this work is the finding that dexmedeto-midine prevents oxidative stress inflammation and celldeath in the neonatal brain under hyperoxic conditionsExposure to high oxygen in the developing brain of six-day-old rat pups leads to increased oxidative stress-induced andinflammatoryDNAdamage as demonstrated by an increasedapoptotic rate a reduction of the GSHGSSG ratio increasedlipid peroxidation and increased IL-1120573 levels Remarkably asingle dose of dexmedetomidine has weakened or abolishedall these detrimental effects under hyperoxic conditionsHowever in this study dexmedetomidine also shows negativeeffects in the control animals and these potential side effectsshould be taken into consideration in further studies on theprotection of the immature brain with dexmedetomidine
Based on these data the hyperoxic model indicatesdexmedetomidine as a sedative in pediatric anesthesia anda possible promising agent for neuroprotective strategies inpreterm infants
Conflict of Interests
The authors declare that there is no conflict of interestswith any financial organization regarding the commercialidentities mentioned in the paper
Acknowledgments
This work was supported by the Forderverein fur fruh-geborene Kinder an der Charite eV Berlin The authorsgratefully thank Evelyn Strauss and Vanessa Valdix for excel-lent technical assistance
References
[1] E Gerdin O Tyden and U J Eriksson ldquoThe developmentof antioxidant enzymatic defense in the perinatal rat lungactivities of superoxide dismutase glutathione peroxidase andcatalaserdquo Pediatric Research vol 19 no 7 pp 687ndash691 1985
[2] R E Black S Cousens H L Johnson et al ldquoGlobal regionaland national causes of child mortality in 2008 a systematicanalysisrdquoThe Lancet vol 375 no 9730 pp 1969ndash1987 2010
[3] M C Allen ldquoNeurodevelopmental outcomes of preterminfantsrdquo Current Opinion in Neurology vol 21 no 2 pp 123ndash128 2008
[4] B R Vohr ldquoNeurodevelopmental outcomes of extremelypreterm infantsrdquo Clinics in Perinatology vol 41 no 1 pp 241ndash255 2014
[5] K J S Anand and R W Hall ldquoPharmacological therapy foranalgesia and sedation in the newbornrdquo Archives of Disease inChildhood Fetal and Neonatal Edition vol 91 no 6 pp F448ndashF453 2006
[6] CMahajan andHHDash ldquoProcedural sedation and analgesiain pediatric patientsrdquo Journal of Pediatric Neurosciences vol 9no 1 pp 1ndash6 2014
[7] S G Soriano andK J S Anand ldquoAnesthetics and brain toxicityrdquoCurrent Opinion in Anaesthesiology vol 18 no 3 pp 293ndash2972005
[8] C McPherson ldquoSedation and analgesia in mechanically ven-tilated preterm neonates continue standard of care or experi-mentrdquoThe Journal of Pediatric Pharmacology andTherapeuticsvol 17 no 4 pp 351ndash364 2012
[9] R Deulofeut D Golde and S Augusto ldquoTreatment-by-gendereffect when aiming to avoid hyperoxia in preterm infants in theNICUrdquo Acta Paediatrica vol 96 no 7 pp 990ndash994 2007
[10] C J Wright and P A Dennery ldquoManipulation of gene expres-sion by oxygen a primer from bedside to benchrdquo PediatricResearch vol 66 no 1 pp 3ndash10 2009
[11] O D Saugstad M Vento S Ramji D Howard and R FSoll ldquoNeurodevelopmental outcome of infants resuscitatedwithair or 100 oxygen a systematic review and meta-analysisrdquoNeonatology vol 102 no 2 pp 98ndash103 2012
[12] U Felderhoff-Mueser P Bittigau M Sifringer et al ldquoOxygencauses cell death in the developing brainrdquo Neurobiology ofDisease vol 17 no 2 pp 273ndash282 2004
[13] M Sifringer K Genz D Brait et al ldquoErythropoietin attenuateshyperoxia-induced cell death by modulation of inflammatorymediators and matrix metalloproteinasesrdquoDevelopmental Neu-roscience vol 31 no 5 pp 394ndash402 2009
[14] M Sifringer D Brait U Weichelt et al ldquoErythropoietinattenuates hyperoxia-induced oxidative stress in the developingrat brainrdquoBrain Behavior and Immunity vol 24 no 5 pp 792ndash799 2010
[15] M Sifringer I Bendix C von Haefen et al ldquoOxygen toxicityis reduced by acetylcholinesterase inhibition in the developingrat brainrdquoDevelopmental Neuroscience vol 35 no 2-3 pp 255ndash264 2013
[16] U Felderhoff-Mueser M Sifringer O Polley et al ldquoCaspase-1-processed interleukins in hyperoxia-induced cell death in thedeveloping brainrdquo Annals of Neurology vol 57 no 1 pp 50ndash592005
[17] S Endesfelder I Zaak U Weichelt C Buhrer and T SchmitzldquoCaffeine protects neuronal cells against injury caused byhyperoxia in the immature brainrdquo Free Radical Biology andMedicine vol 67 pp 221ndash234 2014
[18] F Brehmer I Bendix S Prager et al ldquoInteraction of inflam-mation and hyperoxia in a rat model of neonatal white matterdamagerdquo PLoS ONE vol 7 no 11 Article ID e49023 2012
[19] J Ard W Doyle and A Bekker ldquoAwake craniotomy withdexmedetomidine in pediatric patientsrdquo Journal of Neurosurgi-cal Anesthesiology vol 15 no 3 pp 263ndash266 2003
[20] M A E Ramsay and D L Luterman ldquoDexmedetomidine as atotal intravenous anesthetic agentrdquo Anesthesiology vol 101 no3 pp 787ndash790 2004
[21] S M Walker R F Howard K A Keay and M FitzgeraldldquoDevelopmental age influences the effect of epidural dexmedet-omidine on inflammatory hyperalgesia in rat pupsrdquo Anesthesi-ology vol 102 no 6 pp 1226ndash1234 2005
[22] J Kuhmonen J Pokorny R Miettinen et al ldquoNeuroprotectiveeffects of dexmedetomidine in the gerbil hippocampus aftertransient global ischemiardquoAnesthesiology vol 87 no 2 pp 371ndash377 1997
[23] D Ma M Hossain N Rajakumaraswamy et al ldquoDexmedeto-midine produces its neuroprotective effect via the alpha 2A-adrenoceptor subtyperdquo European Journal of Pharmacology vol502 no 1-2 pp 87ndash97 2004
[24] O Eser H Fidan O Sahin et al ldquoThe influence of dexmedeto-midine on ischemic rat hippocampusrdquo Brain Research vol 1218pp 250ndash256 2008
10 Oxidative Medicine and Cellular Longevity
[25] M Cosar O Eser H Fidan et al ldquoThe neuroprotective effectof dexmedetomidine in the hippocampus of rabbits after sub-arachnoid hemorrhagerdquo Surgical Neurology vol 71 no 1 pp54ndash59 2009
[26] X Duan Y Li C Zhou L Huang and Z Dong ldquoDexmedeto-midine provides neuroprotection impact on ketamine-inducedneuroapoptosis in the developing rat brainrdquo Acta Anaesthesio-logica Scandinavica vol 58 no 9 pp 1121ndash1126 2014
[27] B Xiong Q-Q Shi and C-H Miao ldquoDexmedetomidinerenders a brain protection on hippocampal formation throughinhibition of nNOS-NO signalling in endotoxin-induced shockratsrdquo Brain Injury vol 28 no 7 pp 1003ndash1008 2014
[28] R M Venn J Hell and R M Grounds ldquoRespiratory effectsof dexmedetomidine in the surgical patient requiring intensivecarerdquo Critical Care vol 4 no 5 pp 302ndash308 2000
[29] J D Tobias and J W Berkenbosch ldquoSedation during mechani-cal ventilation in infants and children dexmedetomidine versusmidazolamrdquo Southern Medical Journal vol 97 no 5 pp 451ndash455 2004
[30] J D Tobias ldquoDexmedetomidine to treat opioid withdrawalin infants following prolonged sedation in the pediatric ICUrdquoJournal of Opioid Management vol 2 no 4 pp 201ndash205 2006
[31] R D Sanders J Xu Y Shu et al ldquoDexmedetomidine attenu-ates isoflurane-induced neurocognitive impairment in neonatalratsrdquo Anesthesiology vol 110 no 5 pp 1077ndash1085 2009
[32] R D Sanders P Sun S Patel M Li M Maze and D MaldquoDexmedetomidine provides cortical neuroprotection impacton anaesthetic-induced neuroapoptosis in the rat developingbrainrdquo Acta Anaesthesiologica Scandinavica vol 54 no 6 pp710ndash716 2010
[33] R R Riker Y Shehabi P M Bokesch et al ldquoDexmedetomidinevsmidazolam for sedation of critically Ill patients a randomizedtrialrdquoThe Journal of the American Medical Association vol 301no 5 pp 489ndash499 2009
[34] S M Jakob E Ruokonen R M Grounds et al ldquoDexmedeto-midine vsmidazolamor propofol for sedation during prolongedmechanical ventilation two randomized controlled trialsrdquo TheJournal of the American Medical Association vol 307 no 11 pp1151ndash1160 2012
[35] K OrsquoMara P Gal J L Ransom et al ldquoSuccessful use of dexme-detomidine for sedation in a 24-week gestational age neonaterdquoThe Annals of Pharmacotherapy vol 43 no 10 pp 1707ndash17132009
[36] K OrsquoMara P Gal J Wimmer et al ldquoDexmedetomidine versusstandard therapy with fentanyl for sedation in mechanicallyventilated premature neonatesrdquo The Journal of Pediatric Phar-macology andTherapeutics vol 17 no 3 pp 252ndash262 2012
[37] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[38] U Yis S H Kurul A Kumral et al ldquoHyperoxic exposure leadsto cell death in the developing brainrdquo Brain ampDevelopment vol30 no 9 pp 556ndash562 2008
[39] K Engelhard C Werner S Kaspar et al ldquoEffect of thealpha2-agonist dexmedetomidine on cerebral neurotransmitterconcentrations during cerebral ischemia in ratsrdquoAnesthesiologyvol 96 no 2 pp 450ndash457 2002
[40] Y Li M Zeng W Chen et al ldquoDexmedetomidine reducesisoflurane-induced neuroapoptosis partly by preserving PI3KAkt pathway in the hippocampus of neonatal ratsrdquo PLoS ONEvol 9 no 4 Article ID e93639 2014
[41] AM KaindlM Sifringer A Koppelstaetter et al ldquoErythropoi-etin protects the developing brain from hyperoxia-induced celldeath and proteome changesrdquo Annals of Neurology vol 64 no5 pp 523ndash534 2008
[42] E Koo T Oshodi CMeschter A Ebrahimnejad and G DongldquoNeurotoxic effects of dexmedetomidine in fetal cynomolgusmonkey brainsrdquo Journal of Toxicological Sciences vol 39 no 2pp 251ndash262 2014
[43] H Ayoglu S Gul V Hanci et al ldquoThe effects of dexmedeto-midine dosage on cerebral vasospasm in a rat subarachnoidhaemorrhage modelrdquo Journal of Clinical Neuroscience vol 17no 6 pp 770ndash773 2010
[44] T Nakano and H Okamoto ldquoDexmedetomidine-induced cere-bral hypoperfusion exacerbates ischemic brain injury in ratsrdquoJournal of Anesthesia vol 23 no 3 pp 378ndash384 2009
[45] I Tse H L Zhao and D Q Ma ldquoOrganoprotective effects ofDexmedetomidine from bench to bedsiderdquo Journal of Periop-erative Science vol 1 no 3 pp 1ndash15 2014
[46] M Tasdogan D Memis N Sut and M Yuksel ldquoResults of apilot study on the effects of propofol and dexmedetomidine oninflammatory responses and intraabdominal pressure in severesepsisrdquo Journal of Clinical Anesthesia vol 21 no 6 pp 394ndash4002009
[47] K Tanabe R Matsushima-Nishiwaki O Kozawa and HIida ldquoDexmedetomidine suppresses interleukin-1120573-inducedinterleukin-6 synthesis in rat glial cellsrdquo International Journalof Molecular Medicine vol 34 no 4 pp 1032ndash1038 2014
[48] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitstumor necrosis factor-alpha and interleukin 6 in lipopoly-saccharide-stimulated astrocytes by suppression of c-Jun N-terminal kinasesrdquo Inflammation vol 37 no 3 pp 942ndash9492014
[49] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitsinducible nitric oxide synthase in lipopolysaccharide-stimulat-ed microglia by suppression of extracellular signal-regulatedkinaserdquo Neurological Research vol 37 no 3 pp 238ndash245 2015
[50] C Chrysostomou S R Schulman M H Castellanos et al ldquoAphase IIIII multicenter safety efficacy and pharmacokineticstudy of dexmedetomidine in preterm and term neonatesrdquoTheJournal of Pediatrics vol 164 no 2 pp 276e3ndash282e3 2014
[51] K Konig and K J Guy ldquoBronchopulmonary dysplasia inpreterm infants managed with non-invasive ventilation orsurfactant and a brief period of mechanical ventilation a 6-year cohort studyrdquo The Journal of Maternal-Fetal amp NeonatalMedicine vol 27 no 6 pp 608ndash611 2014
Submit your manuscripts athttpwwwhindawicom
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Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
2 Oxidative Medicine and Cellular Longevity
inhibition of neuronal maturation [17 18] in the developingbrain
Dexmedetomidine is a potent and highly selective agonistof 1205722-receptors with sedative anxiolytic analgesic and anes-thetic properties [19ndash21] In addition it is generally reportedthat dexmedetomidine has neuroprotective effects in differ-ent animal models [22ndash27] and minimal side effects on therespiratory tract and the gastrointestinal function that min-imizes the exposure to other narcotics and benzodiazepines[23 28ndash30] Recent studies suggest the 1205722-adrenoceptor ago-nist dexmedetomidine to attenuate anesthetic agent inducedneuroapoptosis [26 31 32] and it appears for long termsedation as an alternative tomidazolam and propofol [33 34]Advantages of dexmedetomidine are shorter ventilation andrecovery times and lower hypertension and tachycardia Inparticular the shortening of the duration of ventilation seemsto be relevant in connection with the higher oxygen toxicityby prolonged ventilation and the associated development ofchronic lung disease and the consequent motor and cognitivedeficits in preterm born infants First results of clinical trialsin preterm infants revealed a decrease in the duration ofmechanical ventilation by half and no need for additionalsedative administration [35 36]
The aim of this study was to investigate the effect of dif-ferent concentrations of dexmedetomidine to the immaturebrain in a neonatal rat model of oxygen toxicity on neurode-generation oxidative stressmarkers and the expression of theproinflammatory cytokine IL-1120573
2 Materials and Methods
21 Animals and Drug Administration All procedures wereapproved by the State Animal Welfare Authorities (LAGeSoG014513) and followed institutional guidelines Six-day-old Wistar rats from time-pregnant mothers were obtainedfrom Charite-Universitatsmedizin Berlin (Germany) andrandomly assigned to cages and treatment
Dexmedetomidine (DEX dexdor Orion Pharma EspooFinland) was dissolved in phosphate buffered saline Threedoses of the drug (1 5 and 10 120583gkg body weight) wereused and all injections were given intraperitoneally (ip)as a fixed proportion of body weight (100 120583L10 g) The ratpups are divided into different biological groups (descriptionwith the relevant experimental abbreviations) (1) controlgroup (CON 21 O
2 room air) with 09 saline (2) verum
group (21 O2) with 1 120583gkg DEX (DEX1) (3) verum group
with 5 120583gkg DEX (DEX5) (4) verum group with 10 120583gkgDEX (DEX10) (5) hyperoxia group (HY 80 O
2 OxyCycler
BioSpherix Lacona NY) with 09 saline (6) hyperoxiawith 1 120583gkg DEX (HYDEX1) (7) hyperoxia with 5120583gkgDEX (HYDEX5) and (8) hyperoxia with 10 120583gkg DEX(HYDEX10) each with a number of six animals per groupand different gender For hyperoxia or normoxia exposurepups were kept together with their dams Saline or DEXwas administrated once 15min before the start of oxygenexposure
22 Tissue Preparation At 24 h of exposure the animals wereanaesthetized with an ip injection of ketamine (50mgkg)
xylazine (10mgkg) and acepromazine (2mgkg) 5minbefore being perfused For molecular analysis pups weretranscardially perfused with normal saline (pH 74) and thendecapitated the olfactory bulb and cerebellumwere removedand brain hemispheres were snap-frozen in liquid nitrogenand stored at minus80∘C For immunohistochemical analysisanimals were perfused with PBS followed by perfusion with4 paraformaldehyde at pH 74 and the brains were postfixedat 4∘C for 3 days embedded in paraffin and processed forhistological staining
23 DNA Fragmentation Assay Paraffin-embedded sectionswere cut (5 120583m) deparaffinized in Roti-Histol (Carl RothKarlsruhe Germany) twice for 10min each rehydrated indescending ethanol series and rinsed in phosphate bufferedsaline for 3min each at room temperature After deparaf-finization of sections an in situ detection of cells withDNA-strand breaks was performed by the TUNEL labelingmethod using a TdT-FragEL DNA fragmentation detectionkit (Millipore Darmstadt Germany) according to the manu-facturerrsquos instructions Negative controls were performed bysubstituting Tris-buffered saline for the TdT enzyme
The TUNEL positive cells were analyzed in frontal cor-tex (FC) retrosplenial cortex (RSC) hypothalamus (HTH)thalamus (TH) and the hippocampus Sections were viewedby light microscopy while blinded using a LEICA DM 2000microscope equipped with a 200x magnification TUNELpositive cells were counted in the anatomical regions of thebrain in up to twelve different sections per animal and region
24 Determination of Total Glutathione (GSH and GSSG)Total glutathione (GSH and GSSG) was measured in brainhomogenates using the thiol reagent 551015840-dithiobis-2-nitrob-enzoic acid (DTNB) as shown previously [14] In brief for thedetermination of reduced glutathione (GSH) and oxidizedglutathione (GSSG) the brains were homogenized and thehomogenates were treated with a mixture of metaphosphoricacid EDTA and NaCl After centrifugation aliquots weretaken for neutralization with disodium hydrogen phosphatefollowed by addition of DTNB GSH was determined afterreaction with DTNB in a spectrophotometer at 412 nmFor the determination of GSSG 4-vinylpyridine was addedand then incubated for 1 hour at room temperature 4-Vinylpyridine is able to mask the GSH content withoutinterfering with the spectrophotometrical determination ofGSSG at 412 nm GSH and GSSG levels are reported asnmolmg protein
25 Measurement of Lipid Peroxidation Lipid peroxidationwas determined by the reaction of thiobarbituric acid withmalondialdehyde (MDA) a product of lipid breakdowncaused by oxidative stress as previously described [14]A SUPELCOSIL LC-18-DB HPLC reversed-phase column(Sigma-Aldrich Munich Germany 5120583m particle size 250times 10mm ID) was utilized for the detection of MDA levelsin brain homogenates The MDA level was determined byfluorescence (525550 nm) with a 50mM potassium phos-phate buffer (pH 68) and 40 methanol mobile phase at15mLmin flow rate
Oxidative Medicine and Cellular Longevity 3
26 RNA Extraction and Semiquantitative Real-Time PCRTotal RNA was isolated from snap-frozen tissue by acidicphenolchloroform extraction (peqGOLD RNAPurePEQLAB Biotechnologie Erlangen Germany) and 2 120583g ofRNA was reverse-transcribed The PCR products of IL-1120573and hypoxanthine-guanine phosphoribosyltransferase (HPRTas internal standard) were quantified in real time usingdye-labeled fluorogenic reporter oligonucleotide probesand primers (Metabion Munich Germany) with the fol-lowing sequences and corresponding GenBank accessionnumbers IL-1120573 (NM 031512) sense 51015840-AACAAAAAT-GCCTCGTGCTGTCT-31015840 antisense 51015840-TGTTGGCTTATG-TTGTGTCCATTG-31015840 probe 51015840-ACCCATGTGAGCTGA-AAGCTCTCC-31015840 HPRT (NM 012583) sense 51015840-GGAAAG-AACGTCTTGATTGTTGAA-31015840 antisense 51015840-CCAACA-CCTTGAGAGGTCCTTTT-31015840 and probe 51015840-CTTTCC-CTTGGTCAAGCAGTACAGCCCC-31015840 All probes werelabeled at their 51015840 ends with the reporter dye 6-carboxy-fluoresceine (FAM) and at their 31015840 ends with the quencherdye 6-carboxy-tetramethylrhodamine (TAMRA) Real-timePCR and detection were performed in triplicate and repeated3 times for each sample using a total reactive volume of13 120583L which contained 65120583L of 2x TaqMan Universal PCRMaster Mix (Applied Biosystems Foster City CA USA)25 120583L of 125 120583M oligonucleotide mix 05 120583L (05 120583M) ofprobe and 50 ng of cDNA template The PCR amplificationwas performed in 96-well optical reaction plates for 40cycles with each cycle at 94∘C for 15 s and 60∘C for 1minThe expression of IL-1120573 and HPRT was analyzed with thereal-time PCR ABI Prism 7500 sequence detection system(Applied Biosystems) according to the 2minusΔΔCT method [37]
27 Immunoblotting Snap-frozen brain tissue was homog-enized in RIPA buffer solution for protein extraction Thehomogenate was centrifuged at 1050 g (4∘C) for 10min andthe microsomal fraction was subsequently centrifuged at17000 g (4∘C) for 20min After collecting the supernatantprotein concentrations were determined using the PierceBCA kit (Pierce Rockford IL USA) with a 30min incuba-tion at 37∘C prior to spectrophotometry at 562 nm Proteinextracts (30 120583g per sample) were denatured in Laemmlisample loading buffer at 95∘C separated by 15 of sodiumdodecyl sulfate polyacrylamide gel electrophoresis and elec-trotransferred in transfer buffer to a nitrocellulosemembrane(02 120583m pore Protran Schleicher amp Schull Dassel Ger-many) Nonspecific protein binding was prevented by treat-ing the membrane with 5 nonfat dry milk in Tris-bufferedsaline01 Tween 20 for 1 h at room temperature Equalloading and transfer of proteinswas confirmedby staining themembranes with Ponceau S solution (Fluka Buchs Switzer-land) The membranes were incubated overnight at 4∘C withrabbit polyclonal anti-IL-1120573 (17 kDa 1 1000 PromoKineHeidelberg Germany) Horseradish peroxidase-conjugatedsecondary anti-rabbit antibody was diluted 1 2000 (Amer-sham Biosciences Bucks United Kingdom) Positive signalswere visualized using the SuperSignal West Pico kit (Pierce)according to the manufacturerrsquos directions and quantifiedusing a ChemiDoc XRS+ system and the software Image Lab(Bio-RadMunich Germany)Membranes were stripped and
then washed blocked and reprobed overnight at 4∘C withmouse anti-120573-actin monoclonal antibody (42 kDa 1 10000Sigma-Aldrich) Each experiment was repeated three times
28 Statistical Analyses All data are expressed as mean plusmnstandard error of the mean (SEM) Groups were comparedusing a one-way analysis of variance (ANOVA) and sig-nificance was determined using Bonferronirsquos correction formultiple comparisonswith independent sample 119905-test A two-sided 119875 value lt 005 was considered to be significant Allgraphics and statistical analyses were performed using theGraphPad Prism 60 software (GraphPad Software La JollaCA USA)
3 Results
31 Dexmedetomidine Ameliorates Hyperoxia-Induced Neu-rodegeneration in the Infant Brain That oxidative stress isa trigger of cell death is well known We have investigatedthe changes in oxidative stress on the apoptotic cell deathby TUNEL assay These investigations were carried out inthe frontal cortex (FC) and retrosplenial cortex (RSC) inthe deep graymatter (hypothalamus (HTH) thalamus (TH))and in the hippocampus
In detail exposure to 24 h of hyperoxia (HY) from P6 toP7 resulted in a large increase of TUNEL positive cells andthe antiapoptotic ability of dexmedetomidine was able to bedetected by a significant decrease in the investigated differentconcentrations (FC HY 5029 + 130 versus HYDEX12307 + 195 HYDEX5 1550 + 272 and HYDEX10 621+ 72 RSC HY 4483 + 253 versus HYDEX1 1968 +312 HYDEX5 2034 + 246 andHYDEX10 1422 + 259HTH HY 5029 + 450 versus HYDEX1 1426 + 169HYDEX5 1699 + 325 and HYDEX10 1387 + 162 THHY 4424 + 423 versus HYDEX1 1955 + 332 HYDEX52156 + 187 and HYDEX10 1975 + 419) compared tocontrol animals (CON Figures 1(a) and 1(b)) ParticularlyDEX10 under hyperoxic conditions can reduce apoptotic cellrate in the cortex and hypothalamus to control level and sig-nificantly among them in the frontal cortex In the thalamusa significant reduction by DEX under oxygen exposure wasdemonstrated but did not reach the controls DEX undernormoxic conditions showed a significant increase of TUNELpositive cells in the cortices with DEX10 and in the thalamuswith DEX all over (Figure 1(b)) The results of the dentategyrus show a similar antiapoptotic effect of DEX (data notshown) A statistical evaluation of the dentate gyrus was notpossible due to low cell counts
32 Treatment with Dexmedetomidine Modifies Hyperoxia-Affected Levels of Reduced (GSH) and Oxidized Glutathione(GSSG) in the Developing Brain As we showed previ-ously neonatal oxygen toxicity causes an imbalance of theglutathione-redox-system correlating with an increase ofneurodegeneration in the immature brain [12ndash15]
Analysis of GSH and GSSG levels by reaction withthe classical thiol reagent DTNB and spectrophotometricalmeasurement at 412 nmwas performed in samples from totalbrain extracts of 7-day-old rats (119899 = 6 per group) exposed
4 Oxidative Medicine and Cellular Longevity
FC RSC HTH TH
CON
HY
HYDEX1
HYDEX5
HYDEX10
DEX1
DEX10
DEX5
(a)
Figure 1 Continued
Oxidative Medicine and Cellular Longevity 5
Hyperoxia
DEX1
DEX5
DEX10
FC RSC HTH TH0
100
200
300
400
500
600
TUNEL
TUN
EL p
ositi
ve ce
lls (
of c
ontro
l)
+ +
+
+
+
+
+minus
minus
minus
minus
minus minus minus + minus minus
minusminus minus
minusminus
minus
minus + minusminus
minus minus +minus minus
+ +
+
+
+
+
+minus
minus
minus
minus
minus minus minus + minus minus
minusminus minus
minusminus
minus
minus + minusminus
minus minus +minus minus
+ +
+
+
+
+
+minus
minus
minus
minus
minus minus minus + minus minus
minusminus minus
minusminus
minus
minus + minusminus
minus minus +minus minus
+ +
+
+
+
+
+minus
minus
minus
minus
minus minus minus + minus minus
minusminus minus
minusminus
minus
minus + minusminus
minus minus +minus minus
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowastlowast lowastlowastlowastlowast
lowastlowast
lowast lowast
lowastlowast
lowast
(b)
Figure 1 Apoptosis caused by hyperoxia is prevented by dexmedetomidine (a) Representative TUNEL staining images (originalmagnification times400) of rat brain frontal cortices (FC) retrosplenial cortices (RSC) hypothalamus (HTH) and thalamus (TH) of P7 controlpups in room air without (CON) and with dexmedetomidine administration (DEX1 DEX5 and DEX10 corresponding to the concentrationsof 1 5 and 10120583gkg) and after 24 h of hyperoxia from P6 to P7 without (HY) andwith dexmedetomidine administration (HYDEX1 HYDEX5and HYDEX10) (b) Quantitation of TUNEL positive cells in the rat brain frontal cortices (FC) retrosplenial cortices (RSC) hypothalamus(HTH) and thalamus (TH) showed that relative to the control (white bars) hyperoxia at 24 h significantly increased these cell counts in cortexand deep grey matter (black bars) These levels were significantly decreased through systemic dexmedetomidine pretreatment (hatched greybars DEX 1 5 and 10120583gkg) However dexmedetomidine administration resulted in increased TUNEL positive cells in control rats mostprofound in TH (grey bars DEX 1 5 and 10120583gkg) Data are expressed relative to the normoxia-exposed control group (white bars 100)Bars represent mean + SEM 119899 = 6 per group lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus normoxiacontrol 119875 lt 001 and
119875 lt 0001
versus hyperoxia
to (i) normoxia and saline injections (CON) (ii) hyperoxiaand saline injections (HY) (iii) hyperoxia andDEX (HYDEX1 5 or 10 120583gkg) and (iv) normoxia and DEX (DEX 15 or 10 120583gkg) 24 h of hyperoxia triggered the decreaseof GSH levels in brain extracts (Figure 2(a) CON 1142 +011 nmolmg protein and HY 979 + 006 nmolmg protein)When DEX was administered together with hyperoxia itsignificantly increased the levels of GSH at a concentration of5 and 10 120583gkg DEX (HYDEX5 1019 + 006 nmolmg proteinand HYDEX10 1116 + 009 nmolmg protein)
Figure 2(b) shows the increased level of GSSG in thedeveloping brain after 24 h of hyperoxia (CON 0273 +0003 nmolmg protein and HY 0046 + 0007 nmolmgprotein) DEX coapplication reduced the hyperoxia-inducedincrease of GSSG levels significantly at 5 and 10 120583gkg DEX(HYDEX5 0398 + 0008 nmolmg protein and HYDEX100255 + 0008 nmolmg protein)
In total hyperoxia triggered the decrease of the GSHGSSG ratio in the developing rat brain (Figure 2(c) CON4194 + 078 and HY 2152 + 040) When DEX was adminis-tered at the start of hyperoxia the levels of GSHGSSG ratiosignificantly increased at 5 and 10120583gkg of DEX (HYDEX52563 + 060 and HYDEX10 4395 + 162)
33 Induction of Lipid Peroxidation by Hyperoxia in theNeonatal Brain Is Attenuated by Dexmedetomidine TreatmentTo demonstrate that the changes obtained in the level of GSHand GSSG may be connected with an altered lipid perox-idation we examined immature rat brains after treatmentof hyperoxia normoxia dexmedetomidine andor normalsaline treatment for the concentration of malondialdehyde(MDA) Increased MDA levels as a sign of lipid breakdownwere evident in rat brains at 24 h of hyperoxiawhen comparedto normoxic animals whereas a single DEX administrationbefore beginning hyperoxia exposure reduced these levelssignificantly at 5 and 10 120583gkg DEX (Figure 3 CON 563 +014 nmolmg protein and HY 1457 + 016 nmolmg proteinHYDEX5 1362 + 019 nmolmg protein and HYDEX101018 + 031 nmolmg protein)
34 Dexmedetomidine Treatment Reduces IL-1120573 ExpressionunderHyperoxic Conditions in the Infant Brain Aspreviouslyshown by our group hyperoxic conditions lead to an increasein IL-1120573 expression in the immature brain [13 15 16]
Hyperoxia triggered an increase of IL-1120573mRNA in brainhomogenates of neonatal rats as shown by semiquantitativereal-time PCR (Figure 4(a) HY 46599 + 6469) Control
6 Oxidative Medicine and Cellular Longevity
GSH
0123456789
10111213
GSH
(nm
olm
g pr
otei
n)
lowastlowastlowastlowastlowastlowastlowastlowastlowast
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(a)
GSSG
00
01
02
03
04
05
GSS
G (n
mol
mg
prot
ein)
lowastlowastlowastlowastlowastlowast
lowastlowastlowast
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(b)
GSHGSSG ratio
0
10
20
30
40
50
Hyperoxia
DEX1
DEX5
DEX10
GSH
GSS
G ra
tio
lowastlowastlowast lowastlowastlowast
lowastlowastlowast
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(c)
Figure 2 Effect of dexmedetomidine on hyperoxia-modified GSH and GSSG levels in the developing brain (a) Reduced GSH levels wereevident in total rat brain extracts 24 h after the initiation of hyperoxia (black bar) when compared to normoxic animals (white bar) Theselevels were increased through dexmedetomidine (DEX) pretreatment in a concentration dependent manner (hatched grey bars 1 5 and10120583gkg) (b) Increased levels of oxidized GSSG were obvious in total brain extracts at 24 h of hyperoxia (black bar) when compared withnormoxic control animals (white bar)These levels were decreased through pretreatment with DEX (hatched grey bars 1 5 and 10120583gkg) (c)ReducedGSHGSSG ratio levelswere evident in rat brain extracts at 24 h of hyperoxia (black bar)when compared to normoxic control animals(white bar)These levels were upregulated throughDEX pretreatment (hatched grey bars 1 5 and 10120583gkg) Application of dexmedetomidineunder room air (grey bars) showed no effect on GSH or GSSG levels Bars represent mean + SEM 119899 = 6 per group lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 0001 versus hyperoxia
Oxidative Medicine and Cellular Longevity 7
Lipid peroxidation
00
25
50
75
100
125
150
175
MD
A (n
mol
mg
prot
ein)
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
lowastlowastlowast
lowastlowastlowastlowastlowastlowastlowastlowastlowast
lowast
Figure 3 Alteration of lipid peroxidation by hyperoxia in the imma-ture brain Hyperoxia lead to a significant increase of MDA levelsafter 24 h of oxygen exposure (black bar) whereas a single DEXapplication of 5 or 10 120583gkg (hatched grey bars) before hyperoxiaexposure reduced these levels significantly Bars represent mean +SEM 119899 = 6 per group lowast119875 lt 005 and lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 001 and
119875 lt 0001 versus hyperoxia
animals showed low mRNA levels of IL-1120573 (CON 10036 +1141) When dexmedetomidine was administered togetherwith hyperoxia it significantly ameliorated the expression ofproinflammatory IL-1120573 depending on theDEX concentration(HYDEX5 31029 + 2177 and HYDEX10 17766 + 2233)on mRNA levels in rat pups Pretreatment with DEX inanimals kept under normoxic conditions had no effect onIL-1120573 gene expression (DEX1 11718 + 1158 DEX5 12141 +914 and DEX10 13531 + 1285)
Western blotting demonstrated that protein expression ofIL-1120573 is significantly increased following oxygen treatmentfor 24 h (Figure 4(b) HY 33666 + 1725) compared tocontrol animals (CON 10000 + 731) A significant effect onIL-1120573 protein levels was seen upon a single dexmedetomidineapplication of 5 or 10 120583gkg DEX in hyperoxia-exposedanimals (HYDEX5 25096 + 1355 and HYDEX10 17105+ 1227) Animals under normoxic conditions and DEXpretreatment showed low protein levels of IL-1120573 (DEX1 11195+ 443 DEX5 10786 + 515 and DEX10 11244 + 543)
4 Discussion
In the present study we demonstrate that the 1205722-adren-oceptor agonist dexmedetomidine leads to a decrease ofneurodegeneration and affects the level of oxidative stressparameters and the proinflammatory cytokine IL-1120573 in amodel of neonatal hyperoxia-induced brain injury
As shown previously oxygen toxicity leads to an increasedneurodegeneration in the immature brain [12 16 17 38] Inline with these studies here we indicate that an exposure ofsix-day-old rats to a high oxygen concentration (FiO
280)
over 24 h resulted in an increase of neurodegeneration to theneonatal brain (Figures 1(a) and 1(b))Moreover we point outa significant decrease of cell death in the developing rat brainwhen infant rats were treated intraperitoneally with 1 5 or10 120583gkg dexmedetomidine before exposure to high oxygenconditions These dosages are atunder the lower levels ofdexmedetomidine concentrations used in common adult ratmodels of neuronal damage [39 40] and the present dataare not intended to demonstrate the evidence of an effectivedose However our findings conform with different otheranimal studies which examined the neuroprotective capac-ity of dexmedetomidine [22ndash27] Hyperoxia-exposed ratsrevealed an up to 5-fold increase in cell degeneration withinthe cortex and deep gray matter (Figure 1(b)) which hasbeen demonstrated in previous studies [17 41] Interestinglydexmedetomidine treatment showed a profound decrease ofdegenerating cells in all analysed brain regions of rats exposedto hyperoxia (Figure 1(b)) Dexmedetomidine alone inducedno oxidative stress but at a relatively low concentration ofdexmedetomidine under normoxic conditions a significantincrease in apoptosis in the cortex and the thalamus hasbeen shown This result is controversial to other inventions[31 32 42] and to the description of only protective andno toxic effects [24 25 43] In vitro it was shown byKuhmonen et al [22] that dexmedetomidine is more effectiveat lower doses and protects against delayed cell death In anischemia-reperfusionmodel [39] antiapoptotic proteins wereupregulated and proapoptotic proteins are suppressed bydexmedetomidine treatment In the study by Li et al there areno changes in the expression of proapoptotic mediators in anisoflurane-induced model of neuroapoptosis [40] A provenhigh blood pressure after the administration of high-dosedexmedetomidine correlates with cerebral hypoperfusionmay be due to alpha-2-induced cerebral vasoconstriction[44] so that this could contribute to apoptotic cell deathtriggered by other cellular mechanisms However in ourstudy dexmedetomidine also led to negative effects in controlanimals notably in the thalamus and potential side effectsshould be further considered in studies on protection of theimmature brain using dexmedetomidine
The potency of dexmedetomidine in this model of oxy-gen toxicity indicates that the 1205722-adrenoceptor agonist hasantioxidant activities Of note our hyperoxia model is pre-dominantly based on free radical generation as we previouslyfound an induction of different oxidative stress parameters[12ndash15] Therefore we evaluated the effects on biochemicalmarkers of oxidative stress in the developing brain Theconcentrations of GSH and GSSG the GSHGSSG ratio andthe MDA level were used as indicators of oxidative stressand lipid breakdown Hyperoxia-exposed P6 rats showedsignificant decreased GSH and increased GSSG levels at24 h of hyperoxia In addition a decrease in GSHGSSGratio and an increase in MDA level in the developingbrain indicate neuronal damage due to oxidative stressbut there was a modulation of the levels of all parameters
8 Oxidative Medicine and Cellular Longevity
0
100
200
300
400
500
600
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowast
IL-1120573 gene expression
Rela
tive I
L-1120573
gene
expr
essio
n2minusΔΔ
Ct(
)
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(a)
0
100
200
300
400
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
IL-1120573 protein expression
Den
sity
ratio
IL-1120573
120573-a
ctin
( o
f con
trol)
IL-1120573 17kDa
120573-actin 42kDa
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(b)
Figure 4 (a) Quantitative analysis of mRNA expression by real-time PCR showed a marked increase of IL-1120573 mRNA expression in thebrain of P6 rat pups that were kept for 24 h under hyperoxia (black bar) whereas dexmedetomidine treatment restores IL-1120573 upon controllevel (hatched grey bars) depending on the dexmedetomidine concentration Application of dexmedetomidine under room air (grey bars)showed no significant regulation on IL-1120573 mRNA expression (b) The analysis of IL-1120573 protein expression by western blot showed a similarexpression pattern The protein expression of IL-1120573 is significantly increased after 24 h of hyperoxia and a single application of 5 or 10 120583gkgdexmedetomidine could restore the IL-1120573 protein expression almost up to control level The densitometric data represent the ratio of thepixel intensity of the IL-1120573 band to the corresponding 120573-actin band Blots are representative of a series of three blots Data are normalizedto levels of rat pups exposed to normoxia (CON = 100 white bars) Bars represent mean + SEM 119899 = 6 per group lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 005 119875 lt 001 and
119875 lt 0001 versus hyperoxia
up to normoxia levels in hyperoxia-exposed neonatal ratsthat received dexmedetomidine (Figures 2 and 3) Theseobservations suggest that dexmedetomidine seemingly hasan antioxidant activity against hyperoxia-induced oxidativestress in the developing brain In line with our findings thereare several studies that suggest an increased antioxidativecapacity of dexmedetomidine in the brain [24] and furtherorgans (reviewed by Tse et al [45])
This study indicates that dexmedetomidine significantlydecreases hyperoxia-induced IL-1120573 upregulation in the devel-oping rat brainThese results illustrate the protective effect ofdexmedetomidine on neuroinflammation as shown before inclinical and experimental studies [46 47] Dexmedetomidineitself did not affect IL-1120573 levels indicating that the 1205722-adrenoceptor agonist only acts to normalize the inducedproinflammatory cytokine (Figure 4) Here dexmedetomi-dine appears to inhibit the production of inflammatorymediators from multiple cell types for example astrocytesand microglia [48 49]
There are several limitations of this study pointing to areasof future investigations Dexmedetomidine was given onlyonce to the animals while preterm infants receive an initialloading dose and subsequent infusions [50] and the highdoses of dexmedetomidine studied here exceed those dosesrecommended for clinical use short term hyperoxia wasexamined and preterm infants are often exposed to a longerperiod of supraphysiological oxygen concentrations [51] andwe have not determined the plasma levels of dexmedeto-midine Further details of dexmedetomidine-induced sideeffects remain to be elucidated
To our knowledge this is the first report demonstratingthat dexmedetomidine mediates a decrease of neurodegen-eration and oxidative stress and affects the expression of theproinflammatory cytokine IL-1120573 in the hyperoxia-exposedneonatal brain Since oxidative stress and inflammation areinvolved in dysfunction of the immature brain under highoxygen conditions 1205722-adrenoceptor activation seems to bea potential neuroprotective treatment
Oxidative Medicine and Cellular Longevity 9
5 Conclusion
The essential core of this work is the finding that dexmedeto-midine prevents oxidative stress inflammation and celldeath in the neonatal brain under hyperoxic conditionsExposure to high oxygen in the developing brain of six-day-old rat pups leads to increased oxidative stress-induced andinflammatoryDNAdamage as demonstrated by an increasedapoptotic rate a reduction of the GSHGSSG ratio increasedlipid peroxidation and increased IL-1120573 levels Remarkably asingle dose of dexmedetomidine has weakened or abolishedall these detrimental effects under hyperoxic conditionsHowever in this study dexmedetomidine also shows negativeeffects in the control animals and these potential side effectsshould be taken into consideration in further studies on theprotection of the immature brain with dexmedetomidine
Based on these data the hyperoxic model indicatesdexmedetomidine as a sedative in pediatric anesthesia anda possible promising agent for neuroprotective strategies inpreterm infants
Conflict of Interests
The authors declare that there is no conflict of interestswith any financial organization regarding the commercialidentities mentioned in the paper
Acknowledgments
This work was supported by the Forderverein fur fruh-geborene Kinder an der Charite eV Berlin The authorsgratefully thank Evelyn Strauss and Vanessa Valdix for excel-lent technical assistance
References
[1] E Gerdin O Tyden and U J Eriksson ldquoThe developmentof antioxidant enzymatic defense in the perinatal rat lungactivities of superoxide dismutase glutathione peroxidase andcatalaserdquo Pediatric Research vol 19 no 7 pp 687ndash691 1985
[2] R E Black S Cousens H L Johnson et al ldquoGlobal regionaland national causes of child mortality in 2008 a systematicanalysisrdquoThe Lancet vol 375 no 9730 pp 1969ndash1987 2010
[3] M C Allen ldquoNeurodevelopmental outcomes of preterminfantsrdquo Current Opinion in Neurology vol 21 no 2 pp 123ndash128 2008
[4] B R Vohr ldquoNeurodevelopmental outcomes of extremelypreterm infantsrdquo Clinics in Perinatology vol 41 no 1 pp 241ndash255 2014
[5] K J S Anand and R W Hall ldquoPharmacological therapy foranalgesia and sedation in the newbornrdquo Archives of Disease inChildhood Fetal and Neonatal Edition vol 91 no 6 pp F448ndashF453 2006
[6] CMahajan andHHDash ldquoProcedural sedation and analgesiain pediatric patientsrdquo Journal of Pediatric Neurosciences vol 9no 1 pp 1ndash6 2014
[7] S G Soriano andK J S Anand ldquoAnesthetics and brain toxicityrdquoCurrent Opinion in Anaesthesiology vol 18 no 3 pp 293ndash2972005
[8] C McPherson ldquoSedation and analgesia in mechanically ven-tilated preterm neonates continue standard of care or experi-mentrdquoThe Journal of Pediatric Pharmacology andTherapeuticsvol 17 no 4 pp 351ndash364 2012
[9] R Deulofeut D Golde and S Augusto ldquoTreatment-by-gendereffect when aiming to avoid hyperoxia in preterm infants in theNICUrdquo Acta Paediatrica vol 96 no 7 pp 990ndash994 2007
[10] C J Wright and P A Dennery ldquoManipulation of gene expres-sion by oxygen a primer from bedside to benchrdquo PediatricResearch vol 66 no 1 pp 3ndash10 2009
[11] O D Saugstad M Vento S Ramji D Howard and R FSoll ldquoNeurodevelopmental outcome of infants resuscitatedwithair or 100 oxygen a systematic review and meta-analysisrdquoNeonatology vol 102 no 2 pp 98ndash103 2012
[12] U Felderhoff-Mueser P Bittigau M Sifringer et al ldquoOxygencauses cell death in the developing brainrdquo Neurobiology ofDisease vol 17 no 2 pp 273ndash282 2004
[13] M Sifringer K Genz D Brait et al ldquoErythropoietin attenuateshyperoxia-induced cell death by modulation of inflammatorymediators and matrix metalloproteinasesrdquoDevelopmental Neu-roscience vol 31 no 5 pp 394ndash402 2009
[14] M Sifringer D Brait U Weichelt et al ldquoErythropoietinattenuates hyperoxia-induced oxidative stress in the developingrat brainrdquoBrain Behavior and Immunity vol 24 no 5 pp 792ndash799 2010
[15] M Sifringer I Bendix C von Haefen et al ldquoOxygen toxicityis reduced by acetylcholinesterase inhibition in the developingrat brainrdquoDevelopmental Neuroscience vol 35 no 2-3 pp 255ndash264 2013
[16] U Felderhoff-Mueser M Sifringer O Polley et al ldquoCaspase-1-processed interleukins in hyperoxia-induced cell death in thedeveloping brainrdquo Annals of Neurology vol 57 no 1 pp 50ndash592005
[17] S Endesfelder I Zaak U Weichelt C Buhrer and T SchmitzldquoCaffeine protects neuronal cells against injury caused byhyperoxia in the immature brainrdquo Free Radical Biology andMedicine vol 67 pp 221ndash234 2014
[18] F Brehmer I Bendix S Prager et al ldquoInteraction of inflam-mation and hyperoxia in a rat model of neonatal white matterdamagerdquo PLoS ONE vol 7 no 11 Article ID e49023 2012
[19] J Ard W Doyle and A Bekker ldquoAwake craniotomy withdexmedetomidine in pediatric patientsrdquo Journal of Neurosurgi-cal Anesthesiology vol 15 no 3 pp 263ndash266 2003
[20] M A E Ramsay and D L Luterman ldquoDexmedetomidine as atotal intravenous anesthetic agentrdquo Anesthesiology vol 101 no3 pp 787ndash790 2004
[21] S M Walker R F Howard K A Keay and M FitzgeraldldquoDevelopmental age influences the effect of epidural dexmedet-omidine on inflammatory hyperalgesia in rat pupsrdquo Anesthesi-ology vol 102 no 6 pp 1226ndash1234 2005
[22] J Kuhmonen J Pokorny R Miettinen et al ldquoNeuroprotectiveeffects of dexmedetomidine in the gerbil hippocampus aftertransient global ischemiardquoAnesthesiology vol 87 no 2 pp 371ndash377 1997
[23] D Ma M Hossain N Rajakumaraswamy et al ldquoDexmedeto-midine produces its neuroprotective effect via the alpha 2A-adrenoceptor subtyperdquo European Journal of Pharmacology vol502 no 1-2 pp 87ndash97 2004
[24] O Eser H Fidan O Sahin et al ldquoThe influence of dexmedeto-midine on ischemic rat hippocampusrdquo Brain Research vol 1218pp 250ndash256 2008
10 Oxidative Medicine and Cellular Longevity
[25] M Cosar O Eser H Fidan et al ldquoThe neuroprotective effectof dexmedetomidine in the hippocampus of rabbits after sub-arachnoid hemorrhagerdquo Surgical Neurology vol 71 no 1 pp54ndash59 2009
[26] X Duan Y Li C Zhou L Huang and Z Dong ldquoDexmedeto-midine provides neuroprotection impact on ketamine-inducedneuroapoptosis in the developing rat brainrdquo Acta Anaesthesio-logica Scandinavica vol 58 no 9 pp 1121ndash1126 2014
[27] B Xiong Q-Q Shi and C-H Miao ldquoDexmedetomidinerenders a brain protection on hippocampal formation throughinhibition of nNOS-NO signalling in endotoxin-induced shockratsrdquo Brain Injury vol 28 no 7 pp 1003ndash1008 2014
[28] R M Venn J Hell and R M Grounds ldquoRespiratory effectsof dexmedetomidine in the surgical patient requiring intensivecarerdquo Critical Care vol 4 no 5 pp 302ndash308 2000
[29] J D Tobias and J W Berkenbosch ldquoSedation during mechani-cal ventilation in infants and children dexmedetomidine versusmidazolamrdquo Southern Medical Journal vol 97 no 5 pp 451ndash455 2004
[30] J D Tobias ldquoDexmedetomidine to treat opioid withdrawalin infants following prolonged sedation in the pediatric ICUrdquoJournal of Opioid Management vol 2 no 4 pp 201ndash205 2006
[31] R D Sanders J Xu Y Shu et al ldquoDexmedetomidine attenu-ates isoflurane-induced neurocognitive impairment in neonatalratsrdquo Anesthesiology vol 110 no 5 pp 1077ndash1085 2009
[32] R D Sanders P Sun S Patel M Li M Maze and D MaldquoDexmedetomidine provides cortical neuroprotection impacton anaesthetic-induced neuroapoptosis in the rat developingbrainrdquo Acta Anaesthesiologica Scandinavica vol 54 no 6 pp710ndash716 2010
[33] R R Riker Y Shehabi P M Bokesch et al ldquoDexmedetomidinevsmidazolam for sedation of critically Ill patients a randomizedtrialrdquoThe Journal of the American Medical Association vol 301no 5 pp 489ndash499 2009
[34] S M Jakob E Ruokonen R M Grounds et al ldquoDexmedeto-midine vsmidazolamor propofol for sedation during prolongedmechanical ventilation two randomized controlled trialsrdquo TheJournal of the American Medical Association vol 307 no 11 pp1151ndash1160 2012
[35] K OrsquoMara P Gal J L Ransom et al ldquoSuccessful use of dexme-detomidine for sedation in a 24-week gestational age neonaterdquoThe Annals of Pharmacotherapy vol 43 no 10 pp 1707ndash17132009
[36] K OrsquoMara P Gal J Wimmer et al ldquoDexmedetomidine versusstandard therapy with fentanyl for sedation in mechanicallyventilated premature neonatesrdquo The Journal of Pediatric Phar-macology andTherapeutics vol 17 no 3 pp 252ndash262 2012
[37] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[38] U Yis S H Kurul A Kumral et al ldquoHyperoxic exposure leadsto cell death in the developing brainrdquo Brain ampDevelopment vol30 no 9 pp 556ndash562 2008
[39] K Engelhard C Werner S Kaspar et al ldquoEffect of thealpha2-agonist dexmedetomidine on cerebral neurotransmitterconcentrations during cerebral ischemia in ratsrdquoAnesthesiologyvol 96 no 2 pp 450ndash457 2002
[40] Y Li M Zeng W Chen et al ldquoDexmedetomidine reducesisoflurane-induced neuroapoptosis partly by preserving PI3KAkt pathway in the hippocampus of neonatal ratsrdquo PLoS ONEvol 9 no 4 Article ID e93639 2014
[41] AM KaindlM Sifringer A Koppelstaetter et al ldquoErythropoi-etin protects the developing brain from hyperoxia-induced celldeath and proteome changesrdquo Annals of Neurology vol 64 no5 pp 523ndash534 2008
[42] E Koo T Oshodi CMeschter A Ebrahimnejad and G DongldquoNeurotoxic effects of dexmedetomidine in fetal cynomolgusmonkey brainsrdquo Journal of Toxicological Sciences vol 39 no 2pp 251ndash262 2014
[43] H Ayoglu S Gul V Hanci et al ldquoThe effects of dexmedeto-midine dosage on cerebral vasospasm in a rat subarachnoidhaemorrhage modelrdquo Journal of Clinical Neuroscience vol 17no 6 pp 770ndash773 2010
[44] T Nakano and H Okamoto ldquoDexmedetomidine-induced cere-bral hypoperfusion exacerbates ischemic brain injury in ratsrdquoJournal of Anesthesia vol 23 no 3 pp 378ndash384 2009
[45] I Tse H L Zhao and D Q Ma ldquoOrganoprotective effects ofDexmedetomidine from bench to bedsiderdquo Journal of Periop-erative Science vol 1 no 3 pp 1ndash15 2014
[46] M Tasdogan D Memis N Sut and M Yuksel ldquoResults of apilot study on the effects of propofol and dexmedetomidine oninflammatory responses and intraabdominal pressure in severesepsisrdquo Journal of Clinical Anesthesia vol 21 no 6 pp 394ndash4002009
[47] K Tanabe R Matsushima-Nishiwaki O Kozawa and HIida ldquoDexmedetomidine suppresses interleukin-1120573-inducedinterleukin-6 synthesis in rat glial cellsrdquo International Journalof Molecular Medicine vol 34 no 4 pp 1032ndash1038 2014
[48] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitstumor necrosis factor-alpha and interleukin 6 in lipopoly-saccharide-stimulated astrocytes by suppression of c-Jun N-terminal kinasesrdquo Inflammation vol 37 no 3 pp 942ndash9492014
[49] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitsinducible nitric oxide synthase in lipopolysaccharide-stimulat-ed microglia by suppression of extracellular signal-regulatedkinaserdquo Neurological Research vol 37 no 3 pp 238ndash245 2015
[50] C Chrysostomou S R Schulman M H Castellanos et al ldquoAphase IIIII multicenter safety efficacy and pharmacokineticstudy of dexmedetomidine in preterm and term neonatesrdquoTheJournal of Pediatrics vol 164 no 2 pp 276e3ndash282e3 2014
[51] K Konig and K J Guy ldquoBronchopulmonary dysplasia inpreterm infants managed with non-invasive ventilation orsurfactant and a brief period of mechanical ventilation a 6-year cohort studyrdquo The Journal of Maternal-Fetal amp NeonatalMedicine vol 27 no 6 pp 608ndash611 2014
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Oxidative Medicine and Cellular Longevity
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Evidence-Based Complementary and Alternative Medicine
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Oxidative Medicine and Cellular Longevity 3
26 RNA Extraction and Semiquantitative Real-Time PCRTotal RNA was isolated from snap-frozen tissue by acidicphenolchloroform extraction (peqGOLD RNAPurePEQLAB Biotechnologie Erlangen Germany) and 2 120583g ofRNA was reverse-transcribed The PCR products of IL-1120573and hypoxanthine-guanine phosphoribosyltransferase (HPRTas internal standard) were quantified in real time usingdye-labeled fluorogenic reporter oligonucleotide probesand primers (Metabion Munich Germany) with the fol-lowing sequences and corresponding GenBank accessionnumbers IL-1120573 (NM 031512) sense 51015840-AACAAAAAT-GCCTCGTGCTGTCT-31015840 antisense 51015840-TGTTGGCTTATG-TTGTGTCCATTG-31015840 probe 51015840-ACCCATGTGAGCTGA-AAGCTCTCC-31015840 HPRT (NM 012583) sense 51015840-GGAAAG-AACGTCTTGATTGTTGAA-31015840 antisense 51015840-CCAACA-CCTTGAGAGGTCCTTTT-31015840 and probe 51015840-CTTTCC-CTTGGTCAAGCAGTACAGCCCC-31015840 All probes werelabeled at their 51015840 ends with the reporter dye 6-carboxy-fluoresceine (FAM) and at their 31015840 ends with the quencherdye 6-carboxy-tetramethylrhodamine (TAMRA) Real-timePCR and detection were performed in triplicate and repeated3 times for each sample using a total reactive volume of13 120583L which contained 65120583L of 2x TaqMan Universal PCRMaster Mix (Applied Biosystems Foster City CA USA)25 120583L of 125 120583M oligonucleotide mix 05 120583L (05 120583M) ofprobe and 50 ng of cDNA template The PCR amplificationwas performed in 96-well optical reaction plates for 40cycles with each cycle at 94∘C for 15 s and 60∘C for 1minThe expression of IL-1120573 and HPRT was analyzed with thereal-time PCR ABI Prism 7500 sequence detection system(Applied Biosystems) according to the 2minusΔΔCT method [37]
27 Immunoblotting Snap-frozen brain tissue was homog-enized in RIPA buffer solution for protein extraction Thehomogenate was centrifuged at 1050 g (4∘C) for 10min andthe microsomal fraction was subsequently centrifuged at17000 g (4∘C) for 20min After collecting the supernatantprotein concentrations were determined using the PierceBCA kit (Pierce Rockford IL USA) with a 30min incuba-tion at 37∘C prior to spectrophotometry at 562 nm Proteinextracts (30 120583g per sample) were denatured in Laemmlisample loading buffer at 95∘C separated by 15 of sodiumdodecyl sulfate polyacrylamide gel electrophoresis and elec-trotransferred in transfer buffer to a nitrocellulosemembrane(02 120583m pore Protran Schleicher amp Schull Dassel Ger-many) Nonspecific protein binding was prevented by treat-ing the membrane with 5 nonfat dry milk in Tris-bufferedsaline01 Tween 20 for 1 h at room temperature Equalloading and transfer of proteinswas confirmedby staining themembranes with Ponceau S solution (Fluka Buchs Switzer-land) The membranes were incubated overnight at 4∘C withrabbit polyclonal anti-IL-1120573 (17 kDa 1 1000 PromoKineHeidelberg Germany) Horseradish peroxidase-conjugatedsecondary anti-rabbit antibody was diluted 1 2000 (Amer-sham Biosciences Bucks United Kingdom) Positive signalswere visualized using the SuperSignal West Pico kit (Pierce)according to the manufacturerrsquos directions and quantifiedusing a ChemiDoc XRS+ system and the software Image Lab(Bio-RadMunich Germany)Membranes were stripped and
then washed blocked and reprobed overnight at 4∘C withmouse anti-120573-actin monoclonal antibody (42 kDa 1 10000Sigma-Aldrich) Each experiment was repeated three times
28 Statistical Analyses All data are expressed as mean plusmnstandard error of the mean (SEM) Groups were comparedusing a one-way analysis of variance (ANOVA) and sig-nificance was determined using Bonferronirsquos correction formultiple comparisonswith independent sample 119905-test A two-sided 119875 value lt 005 was considered to be significant Allgraphics and statistical analyses were performed using theGraphPad Prism 60 software (GraphPad Software La JollaCA USA)
3 Results
31 Dexmedetomidine Ameliorates Hyperoxia-Induced Neu-rodegeneration in the Infant Brain That oxidative stress isa trigger of cell death is well known We have investigatedthe changes in oxidative stress on the apoptotic cell deathby TUNEL assay These investigations were carried out inthe frontal cortex (FC) and retrosplenial cortex (RSC) inthe deep graymatter (hypothalamus (HTH) thalamus (TH))and in the hippocampus
In detail exposure to 24 h of hyperoxia (HY) from P6 toP7 resulted in a large increase of TUNEL positive cells andthe antiapoptotic ability of dexmedetomidine was able to bedetected by a significant decrease in the investigated differentconcentrations (FC HY 5029 + 130 versus HYDEX12307 + 195 HYDEX5 1550 + 272 and HYDEX10 621+ 72 RSC HY 4483 + 253 versus HYDEX1 1968 +312 HYDEX5 2034 + 246 andHYDEX10 1422 + 259HTH HY 5029 + 450 versus HYDEX1 1426 + 169HYDEX5 1699 + 325 and HYDEX10 1387 + 162 THHY 4424 + 423 versus HYDEX1 1955 + 332 HYDEX52156 + 187 and HYDEX10 1975 + 419) compared tocontrol animals (CON Figures 1(a) and 1(b)) ParticularlyDEX10 under hyperoxic conditions can reduce apoptotic cellrate in the cortex and hypothalamus to control level and sig-nificantly among them in the frontal cortex In the thalamusa significant reduction by DEX under oxygen exposure wasdemonstrated but did not reach the controls DEX undernormoxic conditions showed a significant increase of TUNELpositive cells in the cortices with DEX10 and in the thalamuswith DEX all over (Figure 1(b)) The results of the dentategyrus show a similar antiapoptotic effect of DEX (data notshown) A statistical evaluation of the dentate gyrus was notpossible due to low cell counts
32 Treatment with Dexmedetomidine Modifies Hyperoxia-Affected Levels of Reduced (GSH) and Oxidized Glutathione(GSSG) in the Developing Brain As we showed previ-ously neonatal oxygen toxicity causes an imbalance of theglutathione-redox-system correlating with an increase ofneurodegeneration in the immature brain [12ndash15]
Analysis of GSH and GSSG levels by reaction withthe classical thiol reagent DTNB and spectrophotometricalmeasurement at 412 nmwas performed in samples from totalbrain extracts of 7-day-old rats (119899 = 6 per group) exposed
4 Oxidative Medicine and Cellular Longevity
FC RSC HTH TH
CON
HY
HYDEX1
HYDEX5
HYDEX10
DEX1
DEX10
DEX5
(a)
Figure 1 Continued
Oxidative Medicine and Cellular Longevity 5
Hyperoxia
DEX1
DEX5
DEX10
FC RSC HTH TH0
100
200
300
400
500
600
TUNEL
TUN
EL p
ositi
ve ce
lls (
of c
ontro
l)
+ +
+
+
+
+
+minus
minus
minus
minus
minus minus minus + minus minus
minusminus minus
minusminus
minus
minus + minusminus
minus minus +minus minus
+ +
+
+
+
+
+minus
minus
minus
minus
minus minus minus + minus minus
minusminus minus
minusminus
minus
minus + minusminus
minus minus +minus minus
+ +
+
+
+
+
+minus
minus
minus
minus
minus minus minus + minus minus
minusminus minus
minusminus
minus
minus + minusminus
minus minus +minus minus
+ +
+
+
+
+
+minus
minus
minus
minus
minus minus minus + minus minus
minusminus minus
minusminus
minus
minus + minusminus
minus minus +minus minus
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowastlowast lowastlowastlowastlowast
lowastlowast
lowast lowast
lowastlowast
lowast
(b)
Figure 1 Apoptosis caused by hyperoxia is prevented by dexmedetomidine (a) Representative TUNEL staining images (originalmagnification times400) of rat brain frontal cortices (FC) retrosplenial cortices (RSC) hypothalamus (HTH) and thalamus (TH) of P7 controlpups in room air without (CON) and with dexmedetomidine administration (DEX1 DEX5 and DEX10 corresponding to the concentrationsof 1 5 and 10120583gkg) and after 24 h of hyperoxia from P6 to P7 without (HY) andwith dexmedetomidine administration (HYDEX1 HYDEX5and HYDEX10) (b) Quantitation of TUNEL positive cells in the rat brain frontal cortices (FC) retrosplenial cortices (RSC) hypothalamus(HTH) and thalamus (TH) showed that relative to the control (white bars) hyperoxia at 24 h significantly increased these cell counts in cortexand deep grey matter (black bars) These levels were significantly decreased through systemic dexmedetomidine pretreatment (hatched greybars DEX 1 5 and 10120583gkg) However dexmedetomidine administration resulted in increased TUNEL positive cells in control rats mostprofound in TH (grey bars DEX 1 5 and 10120583gkg) Data are expressed relative to the normoxia-exposed control group (white bars 100)Bars represent mean + SEM 119899 = 6 per group lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus normoxiacontrol 119875 lt 001 and
119875 lt 0001
versus hyperoxia
to (i) normoxia and saline injections (CON) (ii) hyperoxiaand saline injections (HY) (iii) hyperoxia andDEX (HYDEX1 5 or 10 120583gkg) and (iv) normoxia and DEX (DEX 15 or 10 120583gkg) 24 h of hyperoxia triggered the decreaseof GSH levels in brain extracts (Figure 2(a) CON 1142 +011 nmolmg protein and HY 979 + 006 nmolmg protein)When DEX was administered together with hyperoxia itsignificantly increased the levels of GSH at a concentration of5 and 10 120583gkg DEX (HYDEX5 1019 + 006 nmolmg proteinand HYDEX10 1116 + 009 nmolmg protein)
Figure 2(b) shows the increased level of GSSG in thedeveloping brain after 24 h of hyperoxia (CON 0273 +0003 nmolmg protein and HY 0046 + 0007 nmolmgprotein) DEX coapplication reduced the hyperoxia-inducedincrease of GSSG levels significantly at 5 and 10 120583gkg DEX(HYDEX5 0398 + 0008 nmolmg protein and HYDEX100255 + 0008 nmolmg protein)
In total hyperoxia triggered the decrease of the GSHGSSG ratio in the developing rat brain (Figure 2(c) CON4194 + 078 and HY 2152 + 040) When DEX was adminis-tered at the start of hyperoxia the levels of GSHGSSG ratiosignificantly increased at 5 and 10120583gkg of DEX (HYDEX52563 + 060 and HYDEX10 4395 + 162)
33 Induction of Lipid Peroxidation by Hyperoxia in theNeonatal Brain Is Attenuated by Dexmedetomidine TreatmentTo demonstrate that the changes obtained in the level of GSHand GSSG may be connected with an altered lipid perox-idation we examined immature rat brains after treatmentof hyperoxia normoxia dexmedetomidine andor normalsaline treatment for the concentration of malondialdehyde(MDA) Increased MDA levels as a sign of lipid breakdownwere evident in rat brains at 24 h of hyperoxiawhen comparedto normoxic animals whereas a single DEX administrationbefore beginning hyperoxia exposure reduced these levelssignificantly at 5 and 10 120583gkg DEX (Figure 3 CON 563 +014 nmolmg protein and HY 1457 + 016 nmolmg proteinHYDEX5 1362 + 019 nmolmg protein and HYDEX101018 + 031 nmolmg protein)
34 Dexmedetomidine Treatment Reduces IL-1120573 ExpressionunderHyperoxic Conditions in the Infant Brain Aspreviouslyshown by our group hyperoxic conditions lead to an increasein IL-1120573 expression in the immature brain [13 15 16]
Hyperoxia triggered an increase of IL-1120573mRNA in brainhomogenates of neonatal rats as shown by semiquantitativereal-time PCR (Figure 4(a) HY 46599 + 6469) Control
6 Oxidative Medicine and Cellular Longevity
GSH
0123456789
10111213
GSH
(nm
olm
g pr
otei
n)
lowastlowastlowastlowastlowastlowastlowastlowastlowast
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(a)
GSSG
00
01
02
03
04
05
GSS
G (n
mol
mg
prot
ein)
lowastlowastlowastlowastlowastlowast
lowastlowastlowast
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(b)
GSHGSSG ratio
0
10
20
30
40
50
Hyperoxia
DEX1
DEX5
DEX10
GSH
GSS
G ra
tio
lowastlowastlowast lowastlowastlowast
lowastlowastlowast
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(c)
Figure 2 Effect of dexmedetomidine on hyperoxia-modified GSH and GSSG levels in the developing brain (a) Reduced GSH levels wereevident in total rat brain extracts 24 h after the initiation of hyperoxia (black bar) when compared to normoxic animals (white bar) Theselevels were increased through dexmedetomidine (DEX) pretreatment in a concentration dependent manner (hatched grey bars 1 5 and10120583gkg) (b) Increased levels of oxidized GSSG were obvious in total brain extracts at 24 h of hyperoxia (black bar) when compared withnormoxic control animals (white bar)These levels were decreased through pretreatment with DEX (hatched grey bars 1 5 and 10120583gkg) (c)ReducedGSHGSSG ratio levelswere evident in rat brain extracts at 24 h of hyperoxia (black bar)when compared to normoxic control animals(white bar)These levels were upregulated throughDEX pretreatment (hatched grey bars 1 5 and 10120583gkg) Application of dexmedetomidineunder room air (grey bars) showed no effect on GSH or GSSG levels Bars represent mean + SEM 119899 = 6 per group lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 0001 versus hyperoxia
Oxidative Medicine and Cellular Longevity 7
Lipid peroxidation
00
25
50
75
100
125
150
175
MD
A (n
mol
mg
prot
ein)
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
lowastlowastlowast
lowastlowastlowastlowastlowastlowastlowastlowastlowast
lowast
Figure 3 Alteration of lipid peroxidation by hyperoxia in the imma-ture brain Hyperoxia lead to a significant increase of MDA levelsafter 24 h of oxygen exposure (black bar) whereas a single DEXapplication of 5 or 10 120583gkg (hatched grey bars) before hyperoxiaexposure reduced these levels significantly Bars represent mean +SEM 119899 = 6 per group lowast119875 lt 005 and lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 001 and
119875 lt 0001 versus hyperoxia
animals showed low mRNA levels of IL-1120573 (CON 10036 +1141) When dexmedetomidine was administered togetherwith hyperoxia it significantly ameliorated the expression ofproinflammatory IL-1120573 depending on theDEX concentration(HYDEX5 31029 + 2177 and HYDEX10 17766 + 2233)on mRNA levels in rat pups Pretreatment with DEX inanimals kept under normoxic conditions had no effect onIL-1120573 gene expression (DEX1 11718 + 1158 DEX5 12141 +914 and DEX10 13531 + 1285)
Western blotting demonstrated that protein expression ofIL-1120573 is significantly increased following oxygen treatmentfor 24 h (Figure 4(b) HY 33666 + 1725) compared tocontrol animals (CON 10000 + 731) A significant effect onIL-1120573 protein levels was seen upon a single dexmedetomidineapplication of 5 or 10 120583gkg DEX in hyperoxia-exposedanimals (HYDEX5 25096 + 1355 and HYDEX10 17105+ 1227) Animals under normoxic conditions and DEXpretreatment showed low protein levels of IL-1120573 (DEX1 11195+ 443 DEX5 10786 + 515 and DEX10 11244 + 543)
4 Discussion
In the present study we demonstrate that the 1205722-adren-oceptor agonist dexmedetomidine leads to a decrease ofneurodegeneration and affects the level of oxidative stressparameters and the proinflammatory cytokine IL-1120573 in amodel of neonatal hyperoxia-induced brain injury
As shown previously oxygen toxicity leads to an increasedneurodegeneration in the immature brain [12 16 17 38] Inline with these studies here we indicate that an exposure ofsix-day-old rats to a high oxygen concentration (FiO
280)
over 24 h resulted in an increase of neurodegeneration to theneonatal brain (Figures 1(a) and 1(b))Moreover we point outa significant decrease of cell death in the developing rat brainwhen infant rats were treated intraperitoneally with 1 5 or10 120583gkg dexmedetomidine before exposure to high oxygenconditions These dosages are atunder the lower levels ofdexmedetomidine concentrations used in common adult ratmodels of neuronal damage [39 40] and the present dataare not intended to demonstrate the evidence of an effectivedose However our findings conform with different otheranimal studies which examined the neuroprotective capac-ity of dexmedetomidine [22ndash27] Hyperoxia-exposed ratsrevealed an up to 5-fold increase in cell degeneration withinthe cortex and deep gray matter (Figure 1(b)) which hasbeen demonstrated in previous studies [17 41] Interestinglydexmedetomidine treatment showed a profound decrease ofdegenerating cells in all analysed brain regions of rats exposedto hyperoxia (Figure 1(b)) Dexmedetomidine alone inducedno oxidative stress but at a relatively low concentration ofdexmedetomidine under normoxic conditions a significantincrease in apoptosis in the cortex and the thalamus hasbeen shown This result is controversial to other inventions[31 32 42] and to the description of only protective andno toxic effects [24 25 43] In vitro it was shown byKuhmonen et al [22] that dexmedetomidine is more effectiveat lower doses and protects against delayed cell death In anischemia-reperfusionmodel [39] antiapoptotic proteins wereupregulated and proapoptotic proteins are suppressed bydexmedetomidine treatment In the study by Li et al there areno changes in the expression of proapoptotic mediators in anisoflurane-induced model of neuroapoptosis [40] A provenhigh blood pressure after the administration of high-dosedexmedetomidine correlates with cerebral hypoperfusionmay be due to alpha-2-induced cerebral vasoconstriction[44] so that this could contribute to apoptotic cell deathtriggered by other cellular mechanisms However in ourstudy dexmedetomidine also led to negative effects in controlanimals notably in the thalamus and potential side effectsshould be further considered in studies on protection of theimmature brain using dexmedetomidine
The potency of dexmedetomidine in this model of oxy-gen toxicity indicates that the 1205722-adrenoceptor agonist hasantioxidant activities Of note our hyperoxia model is pre-dominantly based on free radical generation as we previouslyfound an induction of different oxidative stress parameters[12ndash15] Therefore we evaluated the effects on biochemicalmarkers of oxidative stress in the developing brain Theconcentrations of GSH and GSSG the GSHGSSG ratio andthe MDA level were used as indicators of oxidative stressand lipid breakdown Hyperoxia-exposed P6 rats showedsignificant decreased GSH and increased GSSG levels at24 h of hyperoxia In addition a decrease in GSHGSSGratio and an increase in MDA level in the developingbrain indicate neuronal damage due to oxidative stressbut there was a modulation of the levels of all parameters
8 Oxidative Medicine and Cellular Longevity
0
100
200
300
400
500
600
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowast
IL-1120573 gene expression
Rela
tive I
L-1120573
gene
expr
essio
n2minusΔΔ
Ct(
)
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(a)
0
100
200
300
400
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
IL-1120573 protein expression
Den
sity
ratio
IL-1120573
120573-a
ctin
( o
f con
trol)
IL-1120573 17kDa
120573-actin 42kDa
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(b)
Figure 4 (a) Quantitative analysis of mRNA expression by real-time PCR showed a marked increase of IL-1120573 mRNA expression in thebrain of P6 rat pups that were kept for 24 h under hyperoxia (black bar) whereas dexmedetomidine treatment restores IL-1120573 upon controllevel (hatched grey bars) depending on the dexmedetomidine concentration Application of dexmedetomidine under room air (grey bars)showed no significant regulation on IL-1120573 mRNA expression (b) The analysis of IL-1120573 protein expression by western blot showed a similarexpression pattern The protein expression of IL-1120573 is significantly increased after 24 h of hyperoxia and a single application of 5 or 10 120583gkgdexmedetomidine could restore the IL-1120573 protein expression almost up to control level The densitometric data represent the ratio of thepixel intensity of the IL-1120573 band to the corresponding 120573-actin band Blots are representative of a series of three blots Data are normalizedto levels of rat pups exposed to normoxia (CON = 100 white bars) Bars represent mean + SEM 119899 = 6 per group lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 005 119875 lt 001 and
119875 lt 0001 versus hyperoxia
up to normoxia levels in hyperoxia-exposed neonatal ratsthat received dexmedetomidine (Figures 2 and 3) Theseobservations suggest that dexmedetomidine seemingly hasan antioxidant activity against hyperoxia-induced oxidativestress in the developing brain In line with our findings thereare several studies that suggest an increased antioxidativecapacity of dexmedetomidine in the brain [24] and furtherorgans (reviewed by Tse et al [45])
This study indicates that dexmedetomidine significantlydecreases hyperoxia-induced IL-1120573 upregulation in the devel-oping rat brainThese results illustrate the protective effect ofdexmedetomidine on neuroinflammation as shown before inclinical and experimental studies [46 47] Dexmedetomidineitself did not affect IL-1120573 levels indicating that the 1205722-adrenoceptor agonist only acts to normalize the inducedproinflammatory cytokine (Figure 4) Here dexmedetomi-dine appears to inhibit the production of inflammatorymediators from multiple cell types for example astrocytesand microglia [48 49]
There are several limitations of this study pointing to areasof future investigations Dexmedetomidine was given onlyonce to the animals while preterm infants receive an initialloading dose and subsequent infusions [50] and the highdoses of dexmedetomidine studied here exceed those dosesrecommended for clinical use short term hyperoxia wasexamined and preterm infants are often exposed to a longerperiod of supraphysiological oxygen concentrations [51] andwe have not determined the plasma levels of dexmedeto-midine Further details of dexmedetomidine-induced sideeffects remain to be elucidated
To our knowledge this is the first report demonstratingthat dexmedetomidine mediates a decrease of neurodegen-eration and oxidative stress and affects the expression of theproinflammatory cytokine IL-1120573 in the hyperoxia-exposedneonatal brain Since oxidative stress and inflammation areinvolved in dysfunction of the immature brain under highoxygen conditions 1205722-adrenoceptor activation seems to bea potential neuroprotective treatment
Oxidative Medicine and Cellular Longevity 9
5 Conclusion
The essential core of this work is the finding that dexmedeto-midine prevents oxidative stress inflammation and celldeath in the neonatal brain under hyperoxic conditionsExposure to high oxygen in the developing brain of six-day-old rat pups leads to increased oxidative stress-induced andinflammatoryDNAdamage as demonstrated by an increasedapoptotic rate a reduction of the GSHGSSG ratio increasedlipid peroxidation and increased IL-1120573 levels Remarkably asingle dose of dexmedetomidine has weakened or abolishedall these detrimental effects under hyperoxic conditionsHowever in this study dexmedetomidine also shows negativeeffects in the control animals and these potential side effectsshould be taken into consideration in further studies on theprotection of the immature brain with dexmedetomidine
Based on these data the hyperoxic model indicatesdexmedetomidine as a sedative in pediatric anesthesia anda possible promising agent for neuroprotective strategies inpreterm infants
Conflict of Interests
The authors declare that there is no conflict of interestswith any financial organization regarding the commercialidentities mentioned in the paper
Acknowledgments
This work was supported by the Forderverein fur fruh-geborene Kinder an der Charite eV Berlin The authorsgratefully thank Evelyn Strauss and Vanessa Valdix for excel-lent technical assistance
References
[1] E Gerdin O Tyden and U J Eriksson ldquoThe developmentof antioxidant enzymatic defense in the perinatal rat lungactivities of superoxide dismutase glutathione peroxidase andcatalaserdquo Pediatric Research vol 19 no 7 pp 687ndash691 1985
[2] R E Black S Cousens H L Johnson et al ldquoGlobal regionaland national causes of child mortality in 2008 a systematicanalysisrdquoThe Lancet vol 375 no 9730 pp 1969ndash1987 2010
[3] M C Allen ldquoNeurodevelopmental outcomes of preterminfantsrdquo Current Opinion in Neurology vol 21 no 2 pp 123ndash128 2008
[4] B R Vohr ldquoNeurodevelopmental outcomes of extremelypreterm infantsrdquo Clinics in Perinatology vol 41 no 1 pp 241ndash255 2014
[5] K J S Anand and R W Hall ldquoPharmacological therapy foranalgesia and sedation in the newbornrdquo Archives of Disease inChildhood Fetal and Neonatal Edition vol 91 no 6 pp F448ndashF453 2006
[6] CMahajan andHHDash ldquoProcedural sedation and analgesiain pediatric patientsrdquo Journal of Pediatric Neurosciences vol 9no 1 pp 1ndash6 2014
[7] S G Soriano andK J S Anand ldquoAnesthetics and brain toxicityrdquoCurrent Opinion in Anaesthesiology vol 18 no 3 pp 293ndash2972005
[8] C McPherson ldquoSedation and analgesia in mechanically ven-tilated preterm neonates continue standard of care or experi-mentrdquoThe Journal of Pediatric Pharmacology andTherapeuticsvol 17 no 4 pp 351ndash364 2012
[9] R Deulofeut D Golde and S Augusto ldquoTreatment-by-gendereffect when aiming to avoid hyperoxia in preterm infants in theNICUrdquo Acta Paediatrica vol 96 no 7 pp 990ndash994 2007
[10] C J Wright and P A Dennery ldquoManipulation of gene expres-sion by oxygen a primer from bedside to benchrdquo PediatricResearch vol 66 no 1 pp 3ndash10 2009
[11] O D Saugstad M Vento S Ramji D Howard and R FSoll ldquoNeurodevelopmental outcome of infants resuscitatedwithair or 100 oxygen a systematic review and meta-analysisrdquoNeonatology vol 102 no 2 pp 98ndash103 2012
[12] U Felderhoff-Mueser P Bittigau M Sifringer et al ldquoOxygencauses cell death in the developing brainrdquo Neurobiology ofDisease vol 17 no 2 pp 273ndash282 2004
[13] M Sifringer K Genz D Brait et al ldquoErythropoietin attenuateshyperoxia-induced cell death by modulation of inflammatorymediators and matrix metalloproteinasesrdquoDevelopmental Neu-roscience vol 31 no 5 pp 394ndash402 2009
[14] M Sifringer D Brait U Weichelt et al ldquoErythropoietinattenuates hyperoxia-induced oxidative stress in the developingrat brainrdquoBrain Behavior and Immunity vol 24 no 5 pp 792ndash799 2010
[15] M Sifringer I Bendix C von Haefen et al ldquoOxygen toxicityis reduced by acetylcholinesterase inhibition in the developingrat brainrdquoDevelopmental Neuroscience vol 35 no 2-3 pp 255ndash264 2013
[16] U Felderhoff-Mueser M Sifringer O Polley et al ldquoCaspase-1-processed interleukins in hyperoxia-induced cell death in thedeveloping brainrdquo Annals of Neurology vol 57 no 1 pp 50ndash592005
[17] S Endesfelder I Zaak U Weichelt C Buhrer and T SchmitzldquoCaffeine protects neuronal cells against injury caused byhyperoxia in the immature brainrdquo Free Radical Biology andMedicine vol 67 pp 221ndash234 2014
[18] F Brehmer I Bendix S Prager et al ldquoInteraction of inflam-mation and hyperoxia in a rat model of neonatal white matterdamagerdquo PLoS ONE vol 7 no 11 Article ID e49023 2012
[19] J Ard W Doyle and A Bekker ldquoAwake craniotomy withdexmedetomidine in pediatric patientsrdquo Journal of Neurosurgi-cal Anesthesiology vol 15 no 3 pp 263ndash266 2003
[20] M A E Ramsay and D L Luterman ldquoDexmedetomidine as atotal intravenous anesthetic agentrdquo Anesthesiology vol 101 no3 pp 787ndash790 2004
[21] S M Walker R F Howard K A Keay and M FitzgeraldldquoDevelopmental age influences the effect of epidural dexmedet-omidine on inflammatory hyperalgesia in rat pupsrdquo Anesthesi-ology vol 102 no 6 pp 1226ndash1234 2005
[22] J Kuhmonen J Pokorny R Miettinen et al ldquoNeuroprotectiveeffects of dexmedetomidine in the gerbil hippocampus aftertransient global ischemiardquoAnesthesiology vol 87 no 2 pp 371ndash377 1997
[23] D Ma M Hossain N Rajakumaraswamy et al ldquoDexmedeto-midine produces its neuroprotective effect via the alpha 2A-adrenoceptor subtyperdquo European Journal of Pharmacology vol502 no 1-2 pp 87ndash97 2004
[24] O Eser H Fidan O Sahin et al ldquoThe influence of dexmedeto-midine on ischemic rat hippocampusrdquo Brain Research vol 1218pp 250ndash256 2008
10 Oxidative Medicine and Cellular Longevity
[25] M Cosar O Eser H Fidan et al ldquoThe neuroprotective effectof dexmedetomidine in the hippocampus of rabbits after sub-arachnoid hemorrhagerdquo Surgical Neurology vol 71 no 1 pp54ndash59 2009
[26] X Duan Y Li C Zhou L Huang and Z Dong ldquoDexmedeto-midine provides neuroprotection impact on ketamine-inducedneuroapoptosis in the developing rat brainrdquo Acta Anaesthesio-logica Scandinavica vol 58 no 9 pp 1121ndash1126 2014
[27] B Xiong Q-Q Shi and C-H Miao ldquoDexmedetomidinerenders a brain protection on hippocampal formation throughinhibition of nNOS-NO signalling in endotoxin-induced shockratsrdquo Brain Injury vol 28 no 7 pp 1003ndash1008 2014
[28] R M Venn J Hell and R M Grounds ldquoRespiratory effectsof dexmedetomidine in the surgical patient requiring intensivecarerdquo Critical Care vol 4 no 5 pp 302ndash308 2000
[29] J D Tobias and J W Berkenbosch ldquoSedation during mechani-cal ventilation in infants and children dexmedetomidine versusmidazolamrdquo Southern Medical Journal vol 97 no 5 pp 451ndash455 2004
[30] J D Tobias ldquoDexmedetomidine to treat opioid withdrawalin infants following prolonged sedation in the pediatric ICUrdquoJournal of Opioid Management vol 2 no 4 pp 201ndash205 2006
[31] R D Sanders J Xu Y Shu et al ldquoDexmedetomidine attenu-ates isoflurane-induced neurocognitive impairment in neonatalratsrdquo Anesthesiology vol 110 no 5 pp 1077ndash1085 2009
[32] R D Sanders P Sun S Patel M Li M Maze and D MaldquoDexmedetomidine provides cortical neuroprotection impacton anaesthetic-induced neuroapoptosis in the rat developingbrainrdquo Acta Anaesthesiologica Scandinavica vol 54 no 6 pp710ndash716 2010
[33] R R Riker Y Shehabi P M Bokesch et al ldquoDexmedetomidinevsmidazolam for sedation of critically Ill patients a randomizedtrialrdquoThe Journal of the American Medical Association vol 301no 5 pp 489ndash499 2009
[34] S M Jakob E Ruokonen R M Grounds et al ldquoDexmedeto-midine vsmidazolamor propofol for sedation during prolongedmechanical ventilation two randomized controlled trialsrdquo TheJournal of the American Medical Association vol 307 no 11 pp1151ndash1160 2012
[35] K OrsquoMara P Gal J L Ransom et al ldquoSuccessful use of dexme-detomidine for sedation in a 24-week gestational age neonaterdquoThe Annals of Pharmacotherapy vol 43 no 10 pp 1707ndash17132009
[36] K OrsquoMara P Gal J Wimmer et al ldquoDexmedetomidine versusstandard therapy with fentanyl for sedation in mechanicallyventilated premature neonatesrdquo The Journal of Pediatric Phar-macology andTherapeutics vol 17 no 3 pp 252ndash262 2012
[37] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[38] U Yis S H Kurul A Kumral et al ldquoHyperoxic exposure leadsto cell death in the developing brainrdquo Brain ampDevelopment vol30 no 9 pp 556ndash562 2008
[39] K Engelhard C Werner S Kaspar et al ldquoEffect of thealpha2-agonist dexmedetomidine on cerebral neurotransmitterconcentrations during cerebral ischemia in ratsrdquoAnesthesiologyvol 96 no 2 pp 450ndash457 2002
[40] Y Li M Zeng W Chen et al ldquoDexmedetomidine reducesisoflurane-induced neuroapoptosis partly by preserving PI3KAkt pathway in the hippocampus of neonatal ratsrdquo PLoS ONEvol 9 no 4 Article ID e93639 2014
[41] AM KaindlM Sifringer A Koppelstaetter et al ldquoErythropoi-etin protects the developing brain from hyperoxia-induced celldeath and proteome changesrdquo Annals of Neurology vol 64 no5 pp 523ndash534 2008
[42] E Koo T Oshodi CMeschter A Ebrahimnejad and G DongldquoNeurotoxic effects of dexmedetomidine in fetal cynomolgusmonkey brainsrdquo Journal of Toxicological Sciences vol 39 no 2pp 251ndash262 2014
[43] H Ayoglu S Gul V Hanci et al ldquoThe effects of dexmedeto-midine dosage on cerebral vasospasm in a rat subarachnoidhaemorrhage modelrdquo Journal of Clinical Neuroscience vol 17no 6 pp 770ndash773 2010
[44] T Nakano and H Okamoto ldquoDexmedetomidine-induced cere-bral hypoperfusion exacerbates ischemic brain injury in ratsrdquoJournal of Anesthesia vol 23 no 3 pp 378ndash384 2009
[45] I Tse H L Zhao and D Q Ma ldquoOrganoprotective effects ofDexmedetomidine from bench to bedsiderdquo Journal of Periop-erative Science vol 1 no 3 pp 1ndash15 2014
[46] M Tasdogan D Memis N Sut and M Yuksel ldquoResults of apilot study on the effects of propofol and dexmedetomidine oninflammatory responses and intraabdominal pressure in severesepsisrdquo Journal of Clinical Anesthesia vol 21 no 6 pp 394ndash4002009
[47] K Tanabe R Matsushima-Nishiwaki O Kozawa and HIida ldquoDexmedetomidine suppresses interleukin-1120573-inducedinterleukin-6 synthesis in rat glial cellsrdquo International Journalof Molecular Medicine vol 34 no 4 pp 1032ndash1038 2014
[48] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitstumor necrosis factor-alpha and interleukin 6 in lipopoly-saccharide-stimulated astrocytes by suppression of c-Jun N-terminal kinasesrdquo Inflammation vol 37 no 3 pp 942ndash9492014
[49] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitsinducible nitric oxide synthase in lipopolysaccharide-stimulat-ed microglia by suppression of extracellular signal-regulatedkinaserdquo Neurological Research vol 37 no 3 pp 238ndash245 2015
[50] C Chrysostomou S R Schulman M H Castellanos et al ldquoAphase IIIII multicenter safety efficacy and pharmacokineticstudy of dexmedetomidine in preterm and term neonatesrdquoTheJournal of Pediatrics vol 164 no 2 pp 276e3ndash282e3 2014
[51] K Konig and K J Guy ldquoBronchopulmonary dysplasia inpreterm infants managed with non-invasive ventilation orsurfactant and a brief period of mechanical ventilation a 6-year cohort studyrdquo The Journal of Maternal-Fetal amp NeonatalMedicine vol 27 no 6 pp 608ndash611 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Diabetes ResearchJournal of
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
4 Oxidative Medicine and Cellular Longevity
FC RSC HTH TH
CON
HY
HYDEX1
HYDEX5
HYDEX10
DEX1
DEX10
DEX5
(a)
Figure 1 Continued
Oxidative Medicine and Cellular Longevity 5
Hyperoxia
DEX1
DEX5
DEX10
FC RSC HTH TH0
100
200
300
400
500
600
TUNEL
TUN
EL p
ositi
ve ce
lls (
of c
ontro
l)
+ +
+
+
+
+
+minus
minus
minus
minus
minus minus minus + minus minus
minusminus minus
minusminus
minus
minus + minusminus
minus minus +minus minus
+ +
+
+
+
+
+minus
minus
minus
minus
minus minus minus + minus minus
minusminus minus
minusminus
minus
minus + minusminus
minus minus +minus minus
+ +
+
+
+
+
+minus
minus
minus
minus
minus minus minus + minus minus
minusminus minus
minusminus
minus
minus + minusminus
minus minus +minus minus
+ +
+
+
+
+
+minus
minus
minus
minus
minus minus minus + minus minus
minusminus minus
minusminus
minus
minus + minusminus
minus minus +minus minus
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowastlowast lowastlowastlowastlowast
lowastlowast
lowast lowast
lowastlowast
lowast
(b)
Figure 1 Apoptosis caused by hyperoxia is prevented by dexmedetomidine (a) Representative TUNEL staining images (originalmagnification times400) of rat brain frontal cortices (FC) retrosplenial cortices (RSC) hypothalamus (HTH) and thalamus (TH) of P7 controlpups in room air without (CON) and with dexmedetomidine administration (DEX1 DEX5 and DEX10 corresponding to the concentrationsof 1 5 and 10120583gkg) and after 24 h of hyperoxia from P6 to P7 without (HY) andwith dexmedetomidine administration (HYDEX1 HYDEX5and HYDEX10) (b) Quantitation of TUNEL positive cells in the rat brain frontal cortices (FC) retrosplenial cortices (RSC) hypothalamus(HTH) and thalamus (TH) showed that relative to the control (white bars) hyperoxia at 24 h significantly increased these cell counts in cortexand deep grey matter (black bars) These levels were significantly decreased through systemic dexmedetomidine pretreatment (hatched greybars DEX 1 5 and 10120583gkg) However dexmedetomidine administration resulted in increased TUNEL positive cells in control rats mostprofound in TH (grey bars DEX 1 5 and 10120583gkg) Data are expressed relative to the normoxia-exposed control group (white bars 100)Bars represent mean + SEM 119899 = 6 per group lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus normoxiacontrol 119875 lt 001 and
119875 lt 0001
versus hyperoxia
to (i) normoxia and saline injections (CON) (ii) hyperoxiaand saline injections (HY) (iii) hyperoxia andDEX (HYDEX1 5 or 10 120583gkg) and (iv) normoxia and DEX (DEX 15 or 10 120583gkg) 24 h of hyperoxia triggered the decreaseof GSH levels in brain extracts (Figure 2(a) CON 1142 +011 nmolmg protein and HY 979 + 006 nmolmg protein)When DEX was administered together with hyperoxia itsignificantly increased the levels of GSH at a concentration of5 and 10 120583gkg DEX (HYDEX5 1019 + 006 nmolmg proteinand HYDEX10 1116 + 009 nmolmg protein)
Figure 2(b) shows the increased level of GSSG in thedeveloping brain after 24 h of hyperoxia (CON 0273 +0003 nmolmg protein and HY 0046 + 0007 nmolmgprotein) DEX coapplication reduced the hyperoxia-inducedincrease of GSSG levels significantly at 5 and 10 120583gkg DEX(HYDEX5 0398 + 0008 nmolmg protein and HYDEX100255 + 0008 nmolmg protein)
In total hyperoxia triggered the decrease of the GSHGSSG ratio in the developing rat brain (Figure 2(c) CON4194 + 078 and HY 2152 + 040) When DEX was adminis-tered at the start of hyperoxia the levels of GSHGSSG ratiosignificantly increased at 5 and 10120583gkg of DEX (HYDEX52563 + 060 and HYDEX10 4395 + 162)
33 Induction of Lipid Peroxidation by Hyperoxia in theNeonatal Brain Is Attenuated by Dexmedetomidine TreatmentTo demonstrate that the changes obtained in the level of GSHand GSSG may be connected with an altered lipid perox-idation we examined immature rat brains after treatmentof hyperoxia normoxia dexmedetomidine andor normalsaline treatment for the concentration of malondialdehyde(MDA) Increased MDA levels as a sign of lipid breakdownwere evident in rat brains at 24 h of hyperoxiawhen comparedto normoxic animals whereas a single DEX administrationbefore beginning hyperoxia exposure reduced these levelssignificantly at 5 and 10 120583gkg DEX (Figure 3 CON 563 +014 nmolmg protein and HY 1457 + 016 nmolmg proteinHYDEX5 1362 + 019 nmolmg protein and HYDEX101018 + 031 nmolmg protein)
34 Dexmedetomidine Treatment Reduces IL-1120573 ExpressionunderHyperoxic Conditions in the Infant Brain Aspreviouslyshown by our group hyperoxic conditions lead to an increasein IL-1120573 expression in the immature brain [13 15 16]
Hyperoxia triggered an increase of IL-1120573mRNA in brainhomogenates of neonatal rats as shown by semiquantitativereal-time PCR (Figure 4(a) HY 46599 + 6469) Control
6 Oxidative Medicine and Cellular Longevity
GSH
0123456789
10111213
GSH
(nm
olm
g pr
otei
n)
lowastlowastlowastlowastlowastlowastlowastlowastlowast
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(a)
GSSG
00
01
02
03
04
05
GSS
G (n
mol
mg
prot
ein)
lowastlowastlowastlowastlowastlowast
lowastlowastlowast
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(b)
GSHGSSG ratio
0
10
20
30
40
50
Hyperoxia
DEX1
DEX5
DEX10
GSH
GSS
G ra
tio
lowastlowastlowast lowastlowastlowast
lowastlowastlowast
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(c)
Figure 2 Effect of dexmedetomidine on hyperoxia-modified GSH and GSSG levels in the developing brain (a) Reduced GSH levels wereevident in total rat brain extracts 24 h after the initiation of hyperoxia (black bar) when compared to normoxic animals (white bar) Theselevels were increased through dexmedetomidine (DEX) pretreatment in a concentration dependent manner (hatched grey bars 1 5 and10120583gkg) (b) Increased levels of oxidized GSSG were obvious in total brain extracts at 24 h of hyperoxia (black bar) when compared withnormoxic control animals (white bar)These levels were decreased through pretreatment with DEX (hatched grey bars 1 5 and 10120583gkg) (c)ReducedGSHGSSG ratio levelswere evident in rat brain extracts at 24 h of hyperoxia (black bar)when compared to normoxic control animals(white bar)These levels were upregulated throughDEX pretreatment (hatched grey bars 1 5 and 10120583gkg) Application of dexmedetomidineunder room air (grey bars) showed no effect on GSH or GSSG levels Bars represent mean + SEM 119899 = 6 per group lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 0001 versus hyperoxia
Oxidative Medicine and Cellular Longevity 7
Lipid peroxidation
00
25
50
75
100
125
150
175
MD
A (n
mol
mg
prot
ein)
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
lowastlowastlowast
lowastlowastlowastlowastlowastlowastlowastlowastlowast
lowast
Figure 3 Alteration of lipid peroxidation by hyperoxia in the imma-ture brain Hyperoxia lead to a significant increase of MDA levelsafter 24 h of oxygen exposure (black bar) whereas a single DEXapplication of 5 or 10 120583gkg (hatched grey bars) before hyperoxiaexposure reduced these levels significantly Bars represent mean +SEM 119899 = 6 per group lowast119875 lt 005 and lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 001 and
119875 lt 0001 versus hyperoxia
animals showed low mRNA levels of IL-1120573 (CON 10036 +1141) When dexmedetomidine was administered togetherwith hyperoxia it significantly ameliorated the expression ofproinflammatory IL-1120573 depending on theDEX concentration(HYDEX5 31029 + 2177 and HYDEX10 17766 + 2233)on mRNA levels in rat pups Pretreatment with DEX inanimals kept under normoxic conditions had no effect onIL-1120573 gene expression (DEX1 11718 + 1158 DEX5 12141 +914 and DEX10 13531 + 1285)
Western blotting demonstrated that protein expression ofIL-1120573 is significantly increased following oxygen treatmentfor 24 h (Figure 4(b) HY 33666 + 1725) compared tocontrol animals (CON 10000 + 731) A significant effect onIL-1120573 protein levels was seen upon a single dexmedetomidineapplication of 5 or 10 120583gkg DEX in hyperoxia-exposedanimals (HYDEX5 25096 + 1355 and HYDEX10 17105+ 1227) Animals under normoxic conditions and DEXpretreatment showed low protein levels of IL-1120573 (DEX1 11195+ 443 DEX5 10786 + 515 and DEX10 11244 + 543)
4 Discussion
In the present study we demonstrate that the 1205722-adren-oceptor agonist dexmedetomidine leads to a decrease ofneurodegeneration and affects the level of oxidative stressparameters and the proinflammatory cytokine IL-1120573 in amodel of neonatal hyperoxia-induced brain injury
As shown previously oxygen toxicity leads to an increasedneurodegeneration in the immature brain [12 16 17 38] Inline with these studies here we indicate that an exposure ofsix-day-old rats to a high oxygen concentration (FiO
280)
over 24 h resulted in an increase of neurodegeneration to theneonatal brain (Figures 1(a) and 1(b))Moreover we point outa significant decrease of cell death in the developing rat brainwhen infant rats were treated intraperitoneally with 1 5 or10 120583gkg dexmedetomidine before exposure to high oxygenconditions These dosages are atunder the lower levels ofdexmedetomidine concentrations used in common adult ratmodels of neuronal damage [39 40] and the present dataare not intended to demonstrate the evidence of an effectivedose However our findings conform with different otheranimal studies which examined the neuroprotective capac-ity of dexmedetomidine [22ndash27] Hyperoxia-exposed ratsrevealed an up to 5-fold increase in cell degeneration withinthe cortex and deep gray matter (Figure 1(b)) which hasbeen demonstrated in previous studies [17 41] Interestinglydexmedetomidine treatment showed a profound decrease ofdegenerating cells in all analysed brain regions of rats exposedto hyperoxia (Figure 1(b)) Dexmedetomidine alone inducedno oxidative stress but at a relatively low concentration ofdexmedetomidine under normoxic conditions a significantincrease in apoptosis in the cortex and the thalamus hasbeen shown This result is controversial to other inventions[31 32 42] and to the description of only protective andno toxic effects [24 25 43] In vitro it was shown byKuhmonen et al [22] that dexmedetomidine is more effectiveat lower doses and protects against delayed cell death In anischemia-reperfusionmodel [39] antiapoptotic proteins wereupregulated and proapoptotic proteins are suppressed bydexmedetomidine treatment In the study by Li et al there areno changes in the expression of proapoptotic mediators in anisoflurane-induced model of neuroapoptosis [40] A provenhigh blood pressure after the administration of high-dosedexmedetomidine correlates with cerebral hypoperfusionmay be due to alpha-2-induced cerebral vasoconstriction[44] so that this could contribute to apoptotic cell deathtriggered by other cellular mechanisms However in ourstudy dexmedetomidine also led to negative effects in controlanimals notably in the thalamus and potential side effectsshould be further considered in studies on protection of theimmature brain using dexmedetomidine
The potency of dexmedetomidine in this model of oxy-gen toxicity indicates that the 1205722-adrenoceptor agonist hasantioxidant activities Of note our hyperoxia model is pre-dominantly based on free radical generation as we previouslyfound an induction of different oxidative stress parameters[12ndash15] Therefore we evaluated the effects on biochemicalmarkers of oxidative stress in the developing brain Theconcentrations of GSH and GSSG the GSHGSSG ratio andthe MDA level were used as indicators of oxidative stressand lipid breakdown Hyperoxia-exposed P6 rats showedsignificant decreased GSH and increased GSSG levels at24 h of hyperoxia In addition a decrease in GSHGSSGratio and an increase in MDA level in the developingbrain indicate neuronal damage due to oxidative stressbut there was a modulation of the levels of all parameters
8 Oxidative Medicine and Cellular Longevity
0
100
200
300
400
500
600
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowast
IL-1120573 gene expression
Rela
tive I
L-1120573
gene
expr
essio
n2minusΔΔ
Ct(
)
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(a)
0
100
200
300
400
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
IL-1120573 protein expression
Den
sity
ratio
IL-1120573
120573-a
ctin
( o
f con
trol)
IL-1120573 17kDa
120573-actin 42kDa
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(b)
Figure 4 (a) Quantitative analysis of mRNA expression by real-time PCR showed a marked increase of IL-1120573 mRNA expression in thebrain of P6 rat pups that were kept for 24 h under hyperoxia (black bar) whereas dexmedetomidine treatment restores IL-1120573 upon controllevel (hatched grey bars) depending on the dexmedetomidine concentration Application of dexmedetomidine under room air (grey bars)showed no significant regulation on IL-1120573 mRNA expression (b) The analysis of IL-1120573 protein expression by western blot showed a similarexpression pattern The protein expression of IL-1120573 is significantly increased after 24 h of hyperoxia and a single application of 5 or 10 120583gkgdexmedetomidine could restore the IL-1120573 protein expression almost up to control level The densitometric data represent the ratio of thepixel intensity of the IL-1120573 band to the corresponding 120573-actin band Blots are representative of a series of three blots Data are normalizedto levels of rat pups exposed to normoxia (CON = 100 white bars) Bars represent mean + SEM 119899 = 6 per group lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 005 119875 lt 001 and
119875 lt 0001 versus hyperoxia
up to normoxia levels in hyperoxia-exposed neonatal ratsthat received dexmedetomidine (Figures 2 and 3) Theseobservations suggest that dexmedetomidine seemingly hasan antioxidant activity against hyperoxia-induced oxidativestress in the developing brain In line with our findings thereare several studies that suggest an increased antioxidativecapacity of dexmedetomidine in the brain [24] and furtherorgans (reviewed by Tse et al [45])
This study indicates that dexmedetomidine significantlydecreases hyperoxia-induced IL-1120573 upregulation in the devel-oping rat brainThese results illustrate the protective effect ofdexmedetomidine on neuroinflammation as shown before inclinical and experimental studies [46 47] Dexmedetomidineitself did not affect IL-1120573 levels indicating that the 1205722-adrenoceptor agonist only acts to normalize the inducedproinflammatory cytokine (Figure 4) Here dexmedetomi-dine appears to inhibit the production of inflammatorymediators from multiple cell types for example astrocytesand microglia [48 49]
There are several limitations of this study pointing to areasof future investigations Dexmedetomidine was given onlyonce to the animals while preterm infants receive an initialloading dose and subsequent infusions [50] and the highdoses of dexmedetomidine studied here exceed those dosesrecommended for clinical use short term hyperoxia wasexamined and preterm infants are often exposed to a longerperiod of supraphysiological oxygen concentrations [51] andwe have not determined the plasma levels of dexmedeto-midine Further details of dexmedetomidine-induced sideeffects remain to be elucidated
To our knowledge this is the first report demonstratingthat dexmedetomidine mediates a decrease of neurodegen-eration and oxidative stress and affects the expression of theproinflammatory cytokine IL-1120573 in the hyperoxia-exposedneonatal brain Since oxidative stress and inflammation areinvolved in dysfunction of the immature brain under highoxygen conditions 1205722-adrenoceptor activation seems to bea potential neuroprotective treatment
Oxidative Medicine and Cellular Longevity 9
5 Conclusion
The essential core of this work is the finding that dexmedeto-midine prevents oxidative stress inflammation and celldeath in the neonatal brain under hyperoxic conditionsExposure to high oxygen in the developing brain of six-day-old rat pups leads to increased oxidative stress-induced andinflammatoryDNAdamage as demonstrated by an increasedapoptotic rate a reduction of the GSHGSSG ratio increasedlipid peroxidation and increased IL-1120573 levels Remarkably asingle dose of dexmedetomidine has weakened or abolishedall these detrimental effects under hyperoxic conditionsHowever in this study dexmedetomidine also shows negativeeffects in the control animals and these potential side effectsshould be taken into consideration in further studies on theprotection of the immature brain with dexmedetomidine
Based on these data the hyperoxic model indicatesdexmedetomidine as a sedative in pediatric anesthesia anda possible promising agent for neuroprotective strategies inpreterm infants
Conflict of Interests
The authors declare that there is no conflict of interestswith any financial organization regarding the commercialidentities mentioned in the paper
Acknowledgments
This work was supported by the Forderverein fur fruh-geborene Kinder an der Charite eV Berlin The authorsgratefully thank Evelyn Strauss and Vanessa Valdix for excel-lent technical assistance
References
[1] E Gerdin O Tyden and U J Eriksson ldquoThe developmentof antioxidant enzymatic defense in the perinatal rat lungactivities of superoxide dismutase glutathione peroxidase andcatalaserdquo Pediatric Research vol 19 no 7 pp 687ndash691 1985
[2] R E Black S Cousens H L Johnson et al ldquoGlobal regionaland national causes of child mortality in 2008 a systematicanalysisrdquoThe Lancet vol 375 no 9730 pp 1969ndash1987 2010
[3] M C Allen ldquoNeurodevelopmental outcomes of preterminfantsrdquo Current Opinion in Neurology vol 21 no 2 pp 123ndash128 2008
[4] B R Vohr ldquoNeurodevelopmental outcomes of extremelypreterm infantsrdquo Clinics in Perinatology vol 41 no 1 pp 241ndash255 2014
[5] K J S Anand and R W Hall ldquoPharmacological therapy foranalgesia and sedation in the newbornrdquo Archives of Disease inChildhood Fetal and Neonatal Edition vol 91 no 6 pp F448ndashF453 2006
[6] CMahajan andHHDash ldquoProcedural sedation and analgesiain pediatric patientsrdquo Journal of Pediatric Neurosciences vol 9no 1 pp 1ndash6 2014
[7] S G Soriano andK J S Anand ldquoAnesthetics and brain toxicityrdquoCurrent Opinion in Anaesthesiology vol 18 no 3 pp 293ndash2972005
[8] C McPherson ldquoSedation and analgesia in mechanically ven-tilated preterm neonates continue standard of care or experi-mentrdquoThe Journal of Pediatric Pharmacology andTherapeuticsvol 17 no 4 pp 351ndash364 2012
[9] R Deulofeut D Golde and S Augusto ldquoTreatment-by-gendereffect when aiming to avoid hyperoxia in preterm infants in theNICUrdquo Acta Paediatrica vol 96 no 7 pp 990ndash994 2007
[10] C J Wright and P A Dennery ldquoManipulation of gene expres-sion by oxygen a primer from bedside to benchrdquo PediatricResearch vol 66 no 1 pp 3ndash10 2009
[11] O D Saugstad M Vento S Ramji D Howard and R FSoll ldquoNeurodevelopmental outcome of infants resuscitatedwithair or 100 oxygen a systematic review and meta-analysisrdquoNeonatology vol 102 no 2 pp 98ndash103 2012
[12] U Felderhoff-Mueser P Bittigau M Sifringer et al ldquoOxygencauses cell death in the developing brainrdquo Neurobiology ofDisease vol 17 no 2 pp 273ndash282 2004
[13] M Sifringer K Genz D Brait et al ldquoErythropoietin attenuateshyperoxia-induced cell death by modulation of inflammatorymediators and matrix metalloproteinasesrdquoDevelopmental Neu-roscience vol 31 no 5 pp 394ndash402 2009
[14] M Sifringer D Brait U Weichelt et al ldquoErythropoietinattenuates hyperoxia-induced oxidative stress in the developingrat brainrdquoBrain Behavior and Immunity vol 24 no 5 pp 792ndash799 2010
[15] M Sifringer I Bendix C von Haefen et al ldquoOxygen toxicityis reduced by acetylcholinesterase inhibition in the developingrat brainrdquoDevelopmental Neuroscience vol 35 no 2-3 pp 255ndash264 2013
[16] U Felderhoff-Mueser M Sifringer O Polley et al ldquoCaspase-1-processed interleukins in hyperoxia-induced cell death in thedeveloping brainrdquo Annals of Neurology vol 57 no 1 pp 50ndash592005
[17] S Endesfelder I Zaak U Weichelt C Buhrer and T SchmitzldquoCaffeine protects neuronal cells against injury caused byhyperoxia in the immature brainrdquo Free Radical Biology andMedicine vol 67 pp 221ndash234 2014
[18] F Brehmer I Bendix S Prager et al ldquoInteraction of inflam-mation and hyperoxia in a rat model of neonatal white matterdamagerdquo PLoS ONE vol 7 no 11 Article ID e49023 2012
[19] J Ard W Doyle and A Bekker ldquoAwake craniotomy withdexmedetomidine in pediatric patientsrdquo Journal of Neurosurgi-cal Anesthesiology vol 15 no 3 pp 263ndash266 2003
[20] M A E Ramsay and D L Luterman ldquoDexmedetomidine as atotal intravenous anesthetic agentrdquo Anesthesiology vol 101 no3 pp 787ndash790 2004
[21] S M Walker R F Howard K A Keay and M FitzgeraldldquoDevelopmental age influences the effect of epidural dexmedet-omidine on inflammatory hyperalgesia in rat pupsrdquo Anesthesi-ology vol 102 no 6 pp 1226ndash1234 2005
[22] J Kuhmonen J Pokorny R Miettinen et al ldquoNeuroprotectiveeffects of dexmedetomidine in the gerbil hippocampus aftertransient global ischemiardquoAnesthesiology vol 87 no 2 pp 371ndash377 1997
[23] D Ma M Hossain N Rajakumaraswamy et al ldquoDexmedeto-midine produces its neuroprotective effect via the alpha 2A-adrenoceptor subtyperdquo European Journal of Pharmacology vol502 no 1-2 pp 87ndash97 2004
[24] O Eser H Fidan O Sahin et al ldquoThe influence of dexmedeto-midine on ischemic rat hippocampusrdquo Brain Research vol 1218pp 250ndash256 2008
10 Oxidative Medicine and Cellular Longevity
[25] M Cosar O Eser H Fidan et al ldquoThe neuroprotective effectof dexmedetomidine in the hippocampus of rabbits after sub-arachnoid hemorrhagerdquo Surgical Neurology vol 71 no 1 pp54ndash59 2009
[26] X Duan Y Li C Zhou L Huang and Z Dong ldquoDexmedeto-midine provides neuroprotection impact on ketamine-inducedneuroapoptosis in the developing rat brainrdquo Acta Anaesthesio-logica Scandinavica vol 58 no 9 pp 1121ndash1126 2014
[27] B Xiong Q-Q Shi and C-H Miao ldquoDexmedetomidinerenders a brain protection on hippocampal formation throughinhibition of nNOS-NO signalling in endotoxin-induced shockratsrdquo Brain Injury vol 28 no 7 pp 1003ndash1008 2014
[28] R M Venn J Hell and R M Grounds ldquoRespiratory effectsof dexmedetomidine in the surgical patient requiring intensivecarerdquo Critical Care vol 4 no 5 pp 302ndash308 2000
[29] J D Tobias and J W Berkenbosch ldquoSedation during mechani-cal ventilation in infants and children dexmedetomidine versusmidazolamrdquo Southern Medical Journal vol 97 no 5 pp 451ndash455 2004
[30] J D Tobias ldquoDexmedetomidine to treat opioid withdrawalin infants following prolonged sedation in the pediatric ICUrdquoJournal of Opioid Management vol 2 no 4 pp 201ndash205 2006
[31] R D Sanders J Xu Y Shu et al ldquoDexmedetomidine attenu-ates isoflurane-induced neurocognitive impairment in neonatalratsrdquo Anesthesiology vol 110 no 5 pp 1077ndash1085 2009
[32] R D Sanders P Sun S Patel M Li M Maze and D MaldquoDexmedetomidine provides cortical neuroprotection impacton anaesthetic-induced neuroapoptosis in the rat developingbrainrdquo Acta Anaesthesiologica Scandinavica vol 54 no 6 pp710ndash716 2010
[33] R R Riker Y Shehabi P M Bokesch et al ldquoDexmedetomidinevsmidazolam for sedation of critically Ill patients a randomizedtrialrdquoThe Journal of the American Medical Association vol 301no 5 pp 489ndash499 2009
[34] S M Jakob E Ruokonen R M Grounds et al ldquoDexmedeto-midine vsmidazolamor propofol for sedation during prolongedmechanical ventilation two randomized controlled trialsrdquo TheJournal of the American Medical Association vol 307 no 11 pp1151ndash1160 2012
[35] K OrsquoMara P Gal J L Ransom et al ldquoSuccessful use of dexme-detomidine for sedation in a 24-week gestational age neonaterdquoThe Annals of Pharmacotherapy vol 43 no 10 pp 1707ndash17132009
[36] K OrsquoMara P Gal J Wimmer et al ldquoDexmedetomidine versusstandard therapy with fentanyl for sedation in mechanicallyventilated premature neonatesrdquo The Journal of Pediatric Phar-macology andTherapeutics vol 17 no 3 pp 252ndash262 2012
[37] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[38] U Yis S H Kurul A Kumral et al ldquoHyperoxic exposure leadsto cell death in the developing brainrdquo Brain ampDevelopment vol30 no 9 pp 556ndash562 2008
[39] K Engelhard C Werner S Kaspar et al ldquoEffect of thealpha2-agonist dexmedetomidine on cerebral neurotransmitterconcentrations during cerebral ischemia in ratsrdquoAnesthesiologyvol 96 no 2 pp 450ndash457 2002
[40] Y Li M Zeng W Chen et al ldquoDexmedetomidine reducesisoflurane-induced neuroapoptosis partly by preserving PI3KAkt pathway in the hippocampus of neonatal ratsrdquo PLoS ONEvol 9 no 4 Article ID e93639 2014
[41] AM KaindlM Sifringer A Koppelstaetter et al ldquoErythropoi-etin protects the developing brain from hyperoxia-induced celldeath and proteome changesrdquo Annals of Neurology vol 64 no5 pp 523ndash534 2008
[42] E Koo T Oshodi CMeschter A Ebrahimnejad and G DongldquoNeurotoxic effects of dexmedetomidine in fetal cynomolgusmonkey brainsrdquo Journal of Toxicological Sciences vol 39 no 2pp 251ndash262 2014
[43] H Ayoglu S Gul V Hanci et al ldquoThe effects of dexmedeto-midine dosage on cerebral vasospasm in a rat subarachnoidhaemorrhage modelrdquo Journal of Clinical Neuroscience vol 17no 6 pp 770ndash773 2010
[44] T Nakano and H Okamoto ldquoDexmedetomidine-induced cere-bral hypoperfusion exacerbates ischemic brain injury in ratsrdquoJournal of Anesthesia vol 23 no 3 pp 378ndash384 2009
[45] I Tse H L Zhao and D Q Ma ldquoOrganoprotective effects ofDexmedetomidine from bench to bedsiderdquo Journal of Periop-erative Science vol 1 no 3 pp 1ndash15 2014
[46] M Tasdogan D Memis N Sut and M Yuksel ldquoResults of apilot study on the effects of propofol and dexmedetomidine oninflammatory responses and intraabdominal pressure in severesepsisrdquo Journal of Clinical Anesthesia vol 21 no 6 pp 394ndash4002009
[47] K Tanabe R Matsushima-Nishiwaki O Kozawa and HIida ldquoDexmedetomidine suppresses interleukin-1120573-inducedinterleukin-6 synthesis in rat glial cellsrdquo International Journalof Molecular Medicine vol 34 no 4 pp 1032ndash1038 2014
[48] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitstumor necrosis factor-alpha and interleukin 6 in lipopoly-saccharide-stimulated astrocytes by suppression of c-Jun N-terminal kinasesrdquo Inflammation vol 37 no 3 pp 942ndash9492014
[49] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitsinducible nitric oxide synthase in lipopolysaccharide-stimulat-ed microglia by suppression of extracellular signal-regulatedkinaserdquo Neurological Research vol 37 no 3 pp 238ndash245 2015
[50] C Chrysostomou S R Schulman M H Castellanos et al ldquoAphase IIIII multicenter safety efficacy and pharmacokineticstudy of dexmedetomidine in preterm and term neonatesrdquoTheJournal of Pediatrics vol 164 no 2 pp 276e3ndash282e3 2014
[51] K Konig and K J Guy ldquoBronchopulmonary dysplasia inpreterm infants managed with non-invasive ventilation orsurfactant and a brief period of mechanical ventilation a 6-year cohort studyrdquo The Journal of Maternal-Fetal amp NeonatalMedicine vol 27 no 6 pp 608ndash611 2014
Submit your manuscripts athttpwwwhindawicom
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
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Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 5
Hyperoxia
DEX1
DEX5
DEX10
FC RSC HTH TH0
100
200
300
400
500
600
TUNEL
TUN
EL p
ositi
ve ce
lls (
of c
ontro
l)
+ +
+
+
+
+
+minus
minus
minus
minus
minus minus minus + minus minus
minusminus minus
minusminus
minus
minus + minusminus
minus minus +minus minus
+ +
+
+
+
+
+minus
minus
minus
minus
minus minus minus + minus minus
minusminus minus
minusminus
minus
minus + minusminus
minus minus +minus minus
+ +
+
+
+
+
+minus
minus
minus
minus
minus minus minus + minus minus
minusminus minus
minusminus
minus
minus + minusminus
minus minus +minus minus
+ +
+
+
+
+
+minus
minus
minus
minus
minus minus minus + minus minus
minusminus minus
minusminus
minus
minus + minusminus
minus minus +minus minus
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowast
lowastlowast lowastlowastlowastlowast
lowastlowast
lowast lowast
lowastlowast
lowast
(b)
Figure 1 Apoptosis caused by hyperoxia is prevented by dexmedetomidine (a) Representative TUNEL staining images (originalmagnification times400) of rat brain frontal cortices (FC) retrosplenial cortices (RSC) hypothalamus (HTH) and thalamus (TH) of P7 controlpups in room air without (CON) and with dexmedetomidine administration (DEX1 DEX5 and DEX10 corresponding to the concentrationsof 1 5 and 10120583gkg) and after 24 h of hyperoxia from P6 to P7 without (HY) andwith dexmedetomidine administration (HYDEX1 HYDEX5and HYDEX10) (b) Quantitation of TUNEL positive cells in the rat brain frontal cortices (FC) retrosplenial cortices (RSC) hypothalamus(HTH) and thalamus (TH) showed that relative to the control (white bars) hyperoxia at 24 h significantly increased these cell counts in cortexand deep grey matter (black bars) These levels were significantly decreased through systemic dexmedetomidine pretreatment (hatched greybars DEX 1 5 and 10120583gkg) However dexmedetomidine administration resulted in increased TUNEL positive cells in control rats mostprofound in TH (grey bars DEX 1 5 and 10120583gkg) Data are expressed relative to the normoxia-exposed control group (white bars 100)Bars represent mean + SEM 119899 = 6 per group lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus normoxiacontrol 119875 lt 001 and
119875 lt 0001
versus hyperoxia
to (i) normoxia and saline injections (CON) (ii) hyperoxiaand saline injections (HY) (iii) hyperoxia andDEX (HYDEX1 5 or 10 120583gkg) and (iv) normoxia and DEX (DEX 15 or 10 120583gkg) 24 h of hyperoxia triggered the decreaseof GSH levels in brain extracts (Figure 2(a) CON 1142 +011 nmolmg protein and HY 979 + 006 nmolmg protein)When DEX was administered together with hyperoxia itsignificantly increased the levels of GSH at a concentration of5 and 10 120583gkg DEX (HYDEX5 1019 + 006 nmolmg proteinand HYDEX10 1116 + 009 nmolmg protein)
Figure 2(b) shows the increased level of GSSG in thedeveloping brain after 24 h of hyperoxia (CON 0273 +0003 nmolmg protein and HY 0046 + 0007 nmolmgprotein) DEX coapplication reduced the hyperoxia-inducedincrease of GSSG levels significantly at 5 and 10 120583gkg DEX(HYDEX5 0398 + 0008 nmolmg protein and HYDEX100255 + 0008 nmolmg protein)
In total hyperoxia triggered the decrease of the GSHGSSG ratio in the developing rat brain (Figure 2(c) CON4194 + 078 and HY 2152 + 040) When DEX was adminis-tered at the start of hyperoxia the levels of GSHGSSG ratiosignificantly increased at 5 and 10120583gkg of DEX (HYDEX52563 + 060 and HYDEX10 4395 + 162)
33 Induction of Lipid Peroxidation by Hyperoxia in theNeonatal Brain Is Attenuated by Dexmedetomidine TreatmentTo demonstrate that the changes obtained in the level of GSHand GSSG may be connected with an altered lipid perox-idation we examined immature rat brains after treatmentof hyperoxia normoxia dexmedetomidine andor normalsaline treatment for the concentration of malondialdehyde(MDA) Increased MDA levels as a sign of lipid breakdownwere evident in rat brains at 24 h of hyperoxiawhen comparedto normoxic animals whereas a single DEX administrationbefore beginning hyperoxia exposure reduced these levelssignificantly at 5 and 10 120583gkg DEX (Figure 3 CON 563 +014 nmolmg protein and HY 1457 + 016 nmolmg proteinHYDEX5 1362 + 019 nmolmg protein and HYDEX101018 + 031 nmolmg protein)
34 Dexmedetomidine Treatment Reduces IL-1120573 ExpressionunderHyperoxic Conditions in the Infant Brain Aspreviouslyshown by our group hyperoxic conditions lead to an increasein IL-1120573 expression in the immature brain [13 15 16]
Hyperoxia triggered an increase of IL-1120573mRNA in brainhomogenates of neonatal rats as shown by semiquantitativereal-time PCR (Figure 4(a) HY 46599 + 6469) Control
6 Oxidative Medicine and Cellular Longevity
GSH
0123456789
10111213
GSH
(nm
olm
g pr
otei
n)
lowastlowastlowastlowastlowastlowastlowastlowastlowast
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(a)
GSSG
00
01
02
03
04
05
GSS
G (n
mol
mg
prot
ein)
lowastlowastlowastlowastlowastlowast
lowastlowastlowast
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(b)
GSHGSSG ratio
0
10
20
30
40
50
Hyperoxia
DEX1
DEX5
DEX10
GSH
GSS
G ra
tio
lowastlowastlowast lowastlowastlowast
lowastlowastlowast
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(c)
Figure 2 Effect of dexmedetomidine on hyperoxia-modified GSH and GSSG levels in the developing brain (a) Reduced GSH levels wereevident in total rat brain extracts 24 h after the initiation of hyperoxia (black bar) when compared to normoxic animals (white bar) Theselevels were increased through dexmedetomidine (DEX) pretreatment in a concentration dependent manner (hatched grey bars 1 5 and10120583gkg) (b) Increased levels of oxidized GSSG were obvious in total brain extracts at 24 h of hyperoxia (black bar) when compared withnormoxic control animals (white bar)These levels were decreased through pretreatment with DEX (hatched grey bars 1 5 and 10120583gkg) (c)ReducedGSHGSSG ratio levelswere evident in rat brain extracts at 24 h of hyperoxia (black bar)when compared to normoxic control animals(white bar)These levels were upregulated throughDEX pretreatment (hatched grey bars 1 5 and 10120583gkg) Application of dexmedetomidineunder room air (grey bars) showed no effect on GSH or GSSG levels Bars represent mean + SEM 119899 = 6 per group lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 0001 versus hyperoxia
Oxidative Medicine and Cellular Longevity 7
Lipid peroxidation
00
25
50
75
100
125
150
175
MD
A (n
mol
mg
prot
ein)
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
lowastlowastlowast
lowastlowastlowastlowastlowastlowastlowastlowastlowast
lowast
Figure 3 Alteration of lipid peroxidation by hyperoxia in the imma-ture brain Hyperoxia lead to a significant increase of MDA levelsafter 24 h of oxygen exposure (black bar) whereas a single DEXapplication of 5 or 10 120583gkg (hatched grey bars) before hyperoxiaexposure reduced these levels significantly Bars represent mean +SEM 119899 = 6 per group lowast119875 lt 005 and lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 001 and
119875 lt 0001 versus hyperoxia
animals showed low mRNA levels of IL-1120573 (CON 10036 +1141) When dexmedetomidine was administered togetherwith hyperoxia it significantly ameliorated the expression ofproinflammatory IL-1120573 depending on theDEX concentration(HYDEX5 31029 + 2177 and HYDEX10 17766 + 2233)on mRNA levels in rat pups Pretreatment with DEX inanimals kept under normoxic conditions had no effect onIL-1120573 gene expression (DEX1 11718 + 1158 DEX5 12141 +914 and DEX10 13531 + 1285)
Western blotting demonstrated that protein expression ofIL-1120573 is significantly increased following oxygen treatmentfor 24 h (Figure 4(b) HY 33666 + 1725) compared tocontrol animals (CON 10000 + 731) A significant effect onIL-1120573 protein levels was seen upon a single dexmedetomidineapplication of 5 or 10 120583gkg DEX in hyperoxia-exposedanimals (HYDEX5 25096 + 1355 and HYDEX10 17105+ 1227) Animals under normoxic conditions and DEXpretreatment showed low protein levels of IL-1120573 (DEX1 11195+ 443 DEX5 10786 + 515 and DEX10 11244 + 543)
4 Discussion
In the present study we demonstrate that the 1205722-adren-oceptor agonist dexmedetomidine leads to a decrease ofneurodegeneration and affects the level of oxidative stressparameters and the proinflammatory cytokine IL-1120573 in amodel of neonatal hyperoxia-induced brain injury
As shown previously oxygen toxicity leads to an increasedneurodegeneration in the immature brain [12 16 17 38] Inline with these studies here we indicate that an exposure ofsix-day-old rats to a high oxygen concentration (FiO
280)
over 24 h resulted in an increase of neurodegeneration to theneonatal brain (Figures 1(a) and 1(b))Moreover we point outa significant decrease of cell death in the developing rat brainwhen infant rats were treated intraperitoneally with 1 5 or10 120583gkg dexmedetomidine before exposure to high oxygenconditions These dosages are atunder the lower levels ofdexmedetomidine concentrations used in common adult ratmodels of neuronal damage [39 40] and the present dataare not intended to demonstrate the evidence of an effectivedose However our findings conform with different otheranimal studies which examined the neuroprotective capac-ity of dexmedetomidine [22ndash27] Hyperoxia-exposed ratsrevealed an up to 5-fold increase in cell degeneration withinthe cortex and deep gray matter (Figure 1(b)) which hasbeen demonstrated in previous studies [17 41] Interestinglydexmedetomidine treatment showed a profound decrease ofdegenerating cells in all analysed brain regions of rats exposedto hyperoxia (Figure 1(b)) Dexmedetomidine alone inducedno oxidative stress but at a relatively low concentration ofdexmedetomidine under normoxic conditions a significantincrease in apoptosis in the cortex and the thalamus hasbeen shown This result is controversial to other inventions[31 32 42] and to the description of only protective andno toxic effects [24 25 43] In vitro it was shown byKuhmonen et al [22] that dexmedetomidine is more effectiveat lower doses and protects against delayed cell death In anischemia-reperfusionmodel [39] antiapoptotic proteins wereupregulated and proapoptotic proteins are suppressed bydexmedetomidine treatment In the study by Li et al there areno changes in the expression of proapoptotic mediators in anisoflurane-induced model of neuroapoptosis [40] A provenhigh blood pressure after the administration of high-dosedexmedetomidine correlates with cerebral hypoperfusionmay be due to alpha-2-induced cerebral vasoconstriction[44] so that this could contribute to apoptotic cell deathtriggered by other cellular mechanisms However in ourstudy dexmedetomidine also led to negative effects in controlanimals notably in the thalamus and potential side effectsshould be further considered in studies on protection of theimmature brain using dexmedetomidine
The potency of dexmedetomidine in this model of oxy-gen toxicity indicates that the 1205722-adrenoceptor agonist hasantioxidant activities Of note our hyperoxia model is pre-dominantly based on free radical generation as we previouslyfound an induction of different oxidative stress parameters[12ndash15] Therefore we evaluated the effects on biochemicalmarkers of oxidative stress in the developing brain Theconcentrations of GSH and GSSG the GSHGSSG ratio andthe MDA level were used as indicators of oxidative stressand lipid breakdown Hyperoxia-exposed P6 rats showedsignificant decreased GSH and increased GSSG levels at24 h of hyperoxia In addition a decrease in GSHGSSGratio and an increase in MDA level in the developingbrain indicate neuronal damage due to oxidative stressbut there was a modulation of the levels of all parameters
8 Oxidative Medicine and Cellular Longevity
0
100
200
300
400
500
600
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowast
IL-1120573 gene expression
Rela
tive I
L-1120573
gene
expr
essio
n2minusΔΔ
Ct(
)
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(a)
0
100
200
300
400
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
IL-1120573 protein expression
Den
sity
ratio
IL-1120573
120573-a
ctin
( o
f con
trol)
IL-1120573 17kDa
120573-actin 42kDa
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(b)
Figure 4 (a) Quantitative analysis of mRNA expression by real-time PCR showed a marked increase of IL-1120573 mRNA expression in thebrain of P6 rat pups that were kept for 24 h under hyperoxia (black bar) whereas dexmedetomidine treatment restores IL-1120573 upon controllevel (hatched grey bars) depending on the dexmedetomidine concentration Application of dexmedetomidine under room air (grey bars)showed no significant regulation on IL-1120573 mRNA expression (b) The analysis of IL-1120573 protein expression by western blot showed a similarexpression pattern The protein expression of IL-1120573 is significantly increased after 24 h of hyperoxia and a single application of 5 or 10 120583gkgdexmedetomidine could restore the IL-1120573 protein expression almost up to control level The densitometric data represent the ratio of thepixel intensity of the IL-1120573 band to the corresponding 120573-actin band Blots are representative of a series of three blots Data are normalizedto levels of rat pups exposed to normoxia (CON = 100 white bars) Bars represent mean + SEM 119899 = 6 per group lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 005 119875 lt 001 and
119875 lt 0001 versus hyperoxia
up to normoxia levels in hyperoxia-exposed neonatal ratsthat received dexmedetomidine (Figures 2 and 3) Theseobservations suggest that dexmedetomidine seemingly hasan antioxidant activity against hyperoxia-induced oxidativestress in the developing brain In line with our findings thereare several studies that suggest an increased antioxidativecapacity of dexmedetomidine in the brain [24] and furtherorgans (reviewed by Tse et al [45])
This study indicates that dexmedetomidine significantlydecreases hyperoxia-induced IL-1120573 upregulation in the devel-oping rat brainThese results illustrate the protective effect ofdexmedetomidine on neuroinflammation as shown before inclinical and experimental studies [46 47] Dexmedetomidineitself did not affect IL-1120573 levels indicating that the 1205722-adrenoceptor agonist only acts to normalize the inducedproinflammatory cytokine (Figure 4) Here dexmedetomi-dine appears to inhibit the production of inflammatorymediators from multiple cell types for example astrocytesand microglia [48 49]
There are several limitations of this study pointing to areasof future investigations Dexmedetomidine was given onlyonce to the animals while preterm infants receive an initialloading dose and subsequent infusions [50] and the highdoses of dexmedetomidine studied here exceed those dosesrecommended for clinical use short term hyperoxia wasexamined and preterm infants are often exposed to a longerperiod of supraphysiological oxygen concentrations [51] andwe have not determined the plasma levels of dexmedeto-midine Further details of dexmedetomidine-induced sideeffects remain to be elucidated
To our knowledge this is the first report demonstratingthat dexmedetomidine mediates a decrease of neurodegen-eration and oxidative stress and affects the expression of theproinflammatory cytokine IL-1120573 in the hyperoxia-exposedneonatal brain Since oxidative stress and inflammation areinvolved in dysfunction of the immature brain under highoxygen conditions 1205722-adrenoceptor activation seems to bea potential neuroprotective treatment
Oxidative Medicine and Cellular Longevity 9
5 Conclusion
The essential core of this work is the finding that dexmedeto-midine prevents oxidative stress inflammation and celldeath in the neonatal brain under hyperoxic conditionsExposure to high oxygen in the developing brain of six-day-old rat pups leads to increased oxidative stress-induced andinflammatoryDNAdamage as demonstrated by an increasedapoptotic rate a reduction of the GSHGSSG ratio increasedlipid peroxidation and increased IL-1120573 levels Remarkably asingle dose of dexmedetomidine has weakened or abolishedall these detrimental effects under hyperoxic conditionsHowever in this study dexmedetomidine also shows negativeeffects in the control animals and these potential side effectsshould be taken into consideration in further studies on theprotection of the immature brain with dexmedetomidine
Based on these data the hyperoxic model indicatesdexmedetomidine as a sedative in pediatric anesthesia anda possible promising agent for neuroprotective strategies inpreterm infants
Conflict of Interests
The authors declare that there is no conflict of interestswith any financial organization regarding the commercialidentities mentioned in the paper
Acknowledgments
This work was supported by the Forderverein fur fruh-geborene Kinder an der Charite eV Berlin The authorsgratefully thank Evelyn Strauss and Vanessa Valdix for excel-lent technical assistance
References
[1] E Gerdin O Tyden and U J Eriksson ldquoThe developmentof antioxidant enzymatic defense in the perinatal rat lungactivities of superoxide dismutase glutathione peroxidase andcatalaserdquo Pediatric Research vol 19 no 7 pp 687ndash691 1985
[2] R E Black S Cousens H L Johnson et al ldquoGlobal regionaland national causes of child mortality in 2008 a systematicanalysisrdquoThe Lancet vol 375 no 9730 pp 1969ndash1987 2010
[3] M C Allen ldquoNeurodevelopmental outcomes of preterminfantsrdquo Current Opinion in Neurology vol 21 no 2 pp 123ndash128 2008
[4] B R Vohr ldquoNeurodevelopmental outcomes of extremelypreterm infantsrdquo Clinics in Perinatology vol 41 no 1 pp 241ndash255 2014
[5] K J S Anand and R W Hall ldquoPharmacological therapy foranalgesia and sedation in the newbornrdquo Archives of Disease inChildhood Fetal and Neonatal Edition vol 91 no 6 pp F448ndashF453 2006
[6] CMahajan andHHDash ldquoProcedural sedation and analgesiain pediatric patientsrdquo Journal of Pediatric Neurosciences vol 9no 1 pp 1ndash6 2014
[7] S G Soriano andK J S Anand ldquoAnesthetics and brain toxicityrdquoCurrent Opinion in Anaesthesiology vol 18 no 3 pp 293ndash2972005
[8] C McPherson ldquoSedation and analgesia in mechanically ven-tilated preterm neonates continue standard of care or experi-mentrdquoThe Journal of Pediatric Pharmacology andTherapeuticsvol 17 no 4 pp 351ndash364 2012
[9] R Deulofeut D Golde and S Augusto ldquoTreatment-by-gendereffect when aiming to avoid hyperoxia in preterm infants in theNICUrdquo Acta Paediatrica vol 96 no 7 pp 990ndash994 2007
[10] C J Wright and P A Dennery ldquoManipulation of gene expres-sion by oxygen a primer from bedside to benchrdquo PediatricResearch vol 66 no 1 pp 3ndash10 2009
[11] O D Saugstad M Vento S Ramji D Howard and R FSoll ldquoNeurodevelopmental outcome of infants resuscitatedwithair or 100 oxygen a systematic review and meta-analysisrdquoNeonatology vol 102 no 2 pp 98ndash103 2012
[12] U Felderhoff-Mueser P Bittigau M Sifringer et al ldquoOxygencauses cell death in the developing brainrdquo Neurobiology ofDisease vol 17 no 2 pp 273ndash282 2004
[13] M Sifringer K Genz D Brait et al ldquoErythropoietin attenuateshyperoxia-induced cell death by modulation of inflammatorymediators and matrix metalloproteinasesrdquoDevelopmental Neu-roscience vol 31 no 5 pp 394ndash402 2009
[14] M Sifringer D Brait U Weichelt et al ldquoErythropoietinattenuates hyperoxia-induced oxidative stress in the developingrat brainrdquoBrain Behavior and Immunity vol 24 no 5 pp 792ndash799 2010
[15] M Sifringer I Bendix C von Haefen et al ldquoOxygen toxicityis reduced by acetylcholinesterase inhibition in the developingrat brainrdquoDevelopmental Neuroscience vol 35 no 2-3 pp 255ndash264 2013
[16] U Felderhoff-Mueser M Sifringer O Polley et al ldquoCaspase-1-processed interleukins in hyperoxia-induced cell death in thedeveloping brainrdquo Annals of Neurology vol 57 no 1 pp 50ndash592005
[17] S Endesfelder I Zaak U Weichelt C Buhrer and T SchmitzldquoCaffeine protects neuronal cells against injury caused byhyperoxia in the immature brainrdquo Free Radical Biology andMedicine vol 67 pp 221ndash234 2014
[18] F Brehmer I Bendix S Prager et al ldquoInteraction of inflam-mation and hyperoxia in a rat model of neonatal white matterdamagerdquo PLoS ONE vol 7 no 11 Article ID e49023 2012
[19] J Ard W Doyle and A Bekker ldquoAwake craniotomy withdexmedetomidine in pediatric patientsrdquo Journal of Neurosurgi-cal Anesthesiology vol 15 no 3 pp 263ndash266 2003
[20] M A E Ramsay and D L Luterman ldquoDexmedetomidine as atotal intravenous anesthetic agentrdquo Anesthesiology vol 101 no3 pp 787ndash790 2004
[21] S M Walker R F Howard K A Keay and M FitzgeraldldquoDevelopmental age influences the effect of epidural dexmedet-omidine on inflammatory hyperalgesia in rat pupsrdquo Anesthesi-ology vol 102 no 6 pp 1226ndash1234 2005
[22] J Kuhmonen J Pokorny R Miettinen et al ldquoNeuroprotectiveeffects of dexmedetomidine in the gerbil hippocampus aftertransient global ischemiardquoAnesthesiology vol 87 no 2 pp 371ndash377 1997
[23] D Ma M Hossain N Rajakumaraswamy et al ldquoDexmedeto-midine produces its neuroprotective effect via the alpha 2A-adrenoceptor subtyperdquo European Journal of Pharmacology vol502 no 1-2 pp 87ndash97 2004
[24] O Eser H Fidan O Sahin et al ldquoThe influence of dexmedeto-midine on ischemic rat hippocampusrdquo Brain Research vol 1218pp 250ndash256 2008
10 Oxidative Medicine and Cellular Longevity
[25] M Cosar O Eser H Fidan et al ldquoThe neuroprotective effectof dexmedetomidine in the hippocampus of rabbits after sub-arachnoid hemorrhagerdquo Surgical Neurology vol 71 no 1 pp54ndash59 2009
[26] X Duan Y Li C Zhou L Huang and Z Dong ldquoDexmedeto-midine provides neuroprotection impact on ketamine-inducedneuroapoptosis in the developing rat brainrdquo Acta Anaesthesio-logica Scandinavica vol 58 no 9 pp 1121ndash1126 2014
[27] B Xiong Q-Q Shi and C-H Miao ldquoDexmedetomidinerenders a brain protection on hippocampal formation throughinhibition of nNOS-NO signalling in endotoxin-induced shockratsrdquo Brain Injury vol 28 no 7 pp 1003ndash1008 2014
[28] R M Venn J Hell and R M Grounds ldquoRespiratory effectsof dexmedetomidine in the surgical patient requiring intensivecarerdquo Critical Care vol 4 no 5 pp 302ndash308 2000
[29] J D Tobias and J W Berkenbosch ldquoSedation during mechani-cal ventilation in infants and children dexmedetomidine versusmidazolamrdquo Southern Medical Journal vol 97 no 5 pp 451ndash455 2004
[30] J D Tobias ldquoDexmedetomidine to treat opioid withdrawalin infants following prolonged sedation in the pediatric ICUrdquoJournal of Opioid Management vol 2 no 4 pp 201ndash205 2006
[31] R D Sanders J Xu Y Shu et al ldquoDexmedetomidine attenu-ates isoflurane-induced neurocognitive impairment in neonatalratsrdquo Anesthesiology vol 110 no 5 pp 1077ndash1085 2009
[32] R D Sanders P Sun S Patel M Li M Maze and D MaldquoDexmedetomidine provides cortical neuroprotection impacton anaesthetic-induced neuroapoptosis in the rat developingbrainrdquo Acta Anaesthesiologica Scandinavica vol 54 no 6 pp710ndash716 2010
[33] R R Riker Y Shehabi P M Bokesch et al ldquoDexmedetomidinevsmidazolam for sedation of critically Ill patients a randomizedtrialrdquoThe Journal of the American Medical Association vol 301no 5 pp 489ndash499 2009
[34] S M Jakob E Ruokonen R M Grounds et al ldquoDexmedeto-midine vsmidazolamor propofol for sedation during prolongedmechanical ventilation two randomized controlled trialsrdquo TheJournal of the American Medical Association vol 307 no 11 pp1151ndash1160 2012
[35] K OrsquoMara P Gal J L Ransom et al ldquoSuccessful use of dexme-detomidine for sedation in a 24-week gestational age neonaterdquoThe Annals of Pharmacotherapy vol 43 no 10 pp 1707ndash17132009
[36] K OrsquoMara P Gal J Wimmer et al ldquoDexmedetomidine versusstandard therapy with fentanyl for sedation in mechanicallyventilated premature neonatesrdquo The Journal of Pediatric Phar-macology andTherapeutics vol 17 no 3 pp 252ndash262 2012
[37] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[38] U Yis S H Kurul A Kumral et al ldquoHyperoxic exposure leadsto cell death in the developing brainrdquo Brain ampDevelopment vol30 no 9 pp 556ndash562 2008
[39] K Engelhard C Werner S Kaspar et al ldquoEffect of thealpha2-agonist dexmedetomidine on cerebral neurotransmitterconcentrations during cerebral ischemia in ratsrdquoAnesthesiologyvol 96 no 2 pp 450ndash457 2002
[40] Y Li M Zeng W Chen et al ldquoDexmedetomidine reducesisoflurane-induced neuroapoptosis partly by preserving PI3KAkt pathway in the hippocampus of neonatal ratsrdquo PLoS ONEvol 9 no 4 Article ID e93639 2014
[41] AM KaindlM Sifringer A Koppelstaetter et al ldquoErythropoi-etin protects the developing brain from hyperoxia-induced celldeath and proteome changesrdquo Annals of Neurology vol 64 no5 pp 523ndash534 2008
[42] E Koo T Oshodi CMeschter A Ebrahimnejad and G DongldquoNeurotoxic effects of dexmedetomidine in fetal cynomolgusmonkey brainsrdquo Journal of Toxicological Sciences vol 39 no 2pp 251ndash262 2014
[43] H Ayoglu S Gul V Hanci et al ldquoThe effects of dexmedeto-midine dosage on cerebral vasospasm in a rat subarachnoidhaemorrhage modelrdquo Journal of Clinical Neuroscience vol 17no 6 pp 770ndash773 2010
[44] T Nakano and H Okamoto ldquoDexmedetomidine-induced cere-bral hypoperfusion exacerbates ischemic brain injury in ratsrdquoJournal of Anesthesia vol 23 no 3 pp 378ndash384 2009
[45] I Tse H L Zhao and D Q Ma ldquoOrganoprotective effects ofDexmedetomidine from bench to bedsiderdquo Journal of Periop-erative Science vol 1 no 3 pp 1ndash15 2014
[46] M Tasdogan D Memis N Sut and M Yuksel ldquoResults of apilot study on the effects of propofol and dexmedetomidine oninflammatory responses and intraabdominal pressure in severesepsisrdquo Journal of Clinical Anesthesia vol 21 no 6 pp 394ndash4002009
[47] K Tanabe R Matsushima-Nishiwaki O Kozawa and HIida ldquoDexmedetomidine suppresses interleukin-1120573-inducedinterleukin-6 synthesis in rat glial cellsrdquo International Journalof Molecular Medicine vol 34 no 4 pp 1032ndash1038 2014
[48] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitstumor necrosis factor-alpha and interleukin 6 in lipopoly-saccharide-stimulated astrocytes by suppression of c-Jun N-terminal kinasesrdquo Inflammation vol 37 no 3 pp 942ndash9492014
[49] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitsinducible nitric oxide synthase in lipopolysaccharide-stimulat-ed microglia by suppression of extracellular signal-regulatedkinaserdquo Neurological Research vol 37 no 3 pp 238ndash245 2015
[50] C Chrysostomou S R Schulman M H Castellanos et al ldquoAphase IIIII multicenter safety efficacy and pharmacokineticstudy of dexmedetomidine in preterm and term neonatesrdquoTheJournal of Pediatrics vol 164 no 2 pp 276e3ndash282e3 2014
[51] K Konig and K J Guy ldquoBronchopulmonary dysplasia inpreterm infants managed with non-invasive ventilation orsurfactant and a brief period of mechanical ventilation a 6-year cohort studyrdquo The Journal of Maternal-Fetal amp NeonatalMedicine vol 27 no 6 pp 608ndash611 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
6 Oxidative Medicine and Cellular Longevity
GSH
0123456789
10111213
GSH
(nm
olm
g pr
otei
n)
lowastlowastlowastlowastlowastlowastlowastlowastlowast
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(a)
GSSG
00
01
02
03
04
05
GSS
G (n
mol
mg
prot
ein)
lowastlowastlowastlowastlowastlowast
lowastlowastlowast
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(b)
GSHGSSG ratio
0
10
20
30
40
50
Hyperoxia
DEX1
DEX5
DEX10
GSH
GSS
G ra
tio
lowastlowastlowast lowastlowastlowast
lowastlowastlowast
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(c)
Figure 2 Effect of dexmedetomidine on hyperoxia-modified GSH and GSSG levels in the developing brain (a) Reduced GSH levels wereevident in total rat brain extracts 24 h after the initiation of hyperoxia (black bar) when compared to normoxic animals (white bar) Theselevels were increased through dexmedetomidine (DEX) pretreatment in a concentration dependent manner (hatched grey bars 1 5 and10120583gkg) (b) Increased levels of oxidized GSSG were obvious in total brain extracts at 24 h of hyperoxia (black bar) when compared withnormoxic control animals (white bar)These levels were decreased through pretreatment with DEX (hatched grey bars 1 5 and 10120583gkg) (c)ReducedGSHGSSG ratio levelswere evident in rat brain extracts at 24 h of hyperoxia (black bar)when compared to normoxic control animals(white bar)These levels were upregulated throughDEX pretreatment (hatched grey bars 1 5 and 10120583gkg) Application of dexmedetomidineunder room air (grey bars) showed no effect on GSH or GSSG levels Bars represent mean + SEM 119899 = 6 per group lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 0001 versus hyperoxia
Oxidative Medicine and Cellular Longevity 7
Lipid peroxidation
00
25
50
75
100
125
150
175
MD
A (n
mol
mg
prot
ein)
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
lowastlowastlowast
lowastlowastlowastlowastlowastlowastlowastlowastlowast
lowast
Figure 3 Alteration of lipid peroxidation by hyperoxia in the imma-ture brain Hyperoxia lead to a significant increase of MDA levelsafter 24 h of oxygen exposure (black bar) whereas a single DEXapplication of 5 or 10 120583gkg (hatched grey bars) before hyperoxiaexposure reduced these levels significantly Bars represent mean +SEM 119899 = 6 per group lowast119875 lt 005 and lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 001 and
119875 lt 0001 versus hyperoxia
animals showed low mRNA levels of IL-1120573 (CON 10036 +1141) When dexmedetomidine was administered togetherwith hyperoxia it significantly ameliorated the expression ofproinflammatory IL-1120573 depending on theDEX concentration(HYDEX5 31029 + 2177 and HYDEX10 17766 + 2233)on mRNA levels in rat pups Pretreatment with DEX inanimals kept under normoxic conditions had no effect onIL-1120573 gene expression (DEX1 11718 + 1158 DEX5 12141 +914 and DEX10 13531 + 1285)
Western blotting demonstrated that protein expression ofIL-1120573 is significantly increased following oxygen treatmentfor 24 h (Figure 4(b) HY 33666 + 1725) compared tocontrol animals (CON 10000 + 731) A significant effect onIL-1120573 protein levels was seen upon a single dexmedetomidineapplication of 5 or 10 120583gkg DEX in hyperoxia-exposedanimals (HYDEX5 25096 + 1355 and HYDEX10 17105+ 1227) Animals under normoxic conditions and DEXpretreatment showed low protein levels of IL-1120573 (DEX1 11195+ 443 DEX5 10786 + 515 and DEX10 11244 + 543)
4 Discussion
In the present study we demonstrate that the 1205722-adren-oceptor agonist dexmedetomidine leads to a decrease ofneurodegeneration and affects the level of oxidative stressparameters and the proinflammatory cytokine IL-1120573 in amodel of neonatal hyperoxia-induced brain injury
As shown previously oxygen toxicity leads to an increasedneurodegeneration in the immature brain [12 16 17 38] Inline with these studies here we indicate that an exposure ofsix-day-old rats to a high oxygen concentration (FiO
280)
over 24 h resulted in an increase of neurodegeneration to theneonatal brain (Figures 1(a) and 1(b))Moreover we point outa significant decrease of cell death in the developing rat brainwhen infant rats were treated intraperitoneally with 1 5 or10 120583gkg dexmedetomidine before exposure to high oxygenconditions These dosages are atunder the lower levels ofdexmedetomidine concentrations used in common adult ratmodels of neuronal damage [39 40] and the present dataare not intended to demonstrate the evidence of an effectivedose However our findings conform with different otheranimal studies which examined the neuroprotective capac-ity of dexmedetomidine [22ndash27] Hyperoxia-exposed ratsrevealed an up to 5-fold increase in cell degeneration withinthe cortex and deep gray matter (Figure 1(b)) which hasbeen demonstrated in previous studies [17 41] Interestinglydexmedetomidine treatment showed a profound decrease ofdegenerating cells in all analysed brain regions of rats exposedto hyperoxia (Figure 1(b)) Dexmedetomidine alone inducedno oxidative stress but at a relatively low concentration ofdexmedetomidine under normoxic conditions a significantincrease in apoptosis in the cortex and the thalamus hasbeen shown This result is controversial to other inventions[31 32 42] and to the description of only protective andno toxic effects [24 25 43] In vitro it was shown byKuhmonen et al [22] that dexmedetomidine is more effectiveat lower doses and protects against delayed cell death In anischemia-reperfusionmodel [39] antiapoptotic proteins wereupregulated and proapoptotic proteins are suppressed bydexmedetomidine treatment In the study by Li et al there areno changes in the expression of proapoptotic mediators in anisoflurane-induced model of neuroapoptosis [40] A provenhigh blood pressure after the administration of high-dosedexmedetomidine correlates with cerebral hypoperfusionmay be due to alpha-2-induced cerebral vasoconstriction[44] so that this could contribute to apoptotic cell deathtriggered by other cellular mechanisms However in ourstudy dexmedetomidine also led to negative effects in controlanimals notably in the thalamus and potential side effectsshould be further considered in studies on protection of theimmature brain using dexmedetomidine
The potency of dexmedetomidine in this model of oxy-gen toxicity indicates that the 1205722-adrenoceptor agonist hasantioxidant activities Of note our hyperoxia model is pre-dominantly based on free radical generation as we previouslyfound an induction of different oxidative stress parameters[12ndash15] Therefore we evaluated the effects on biochemicalmarkers of oxidative stress in the developing brain Theconcentrations of GSH and GSSG the GSHGSSG ratio andthe MDA level were used as indicators of oxidative stressand lipid breakdown Hyperoxia-exposed P6 rats showedsignificant decreased GSH and increased GSSG levels at24 h of hyperoxia In addition a decrease in GSHGSSGratio and an increase in MDA level in the developingbrain indicate neuronal damage due to oxidative stressbut there was a modulation of the levels of all parameters
8 Oxidative Medicine and Cellular Longevity
0
100
200
300
400
500
600
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowast
IL-1120573 gene expression
Rela
tive I
L-1120573
gene
expr
essio
n2minusΔΔ
Ct(
)
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(a)
0
100
200
300
400
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
IL-1120573 protein expression
Den
sity
ratio
IL-1120573
120573-a
ctin
( o
f con
trol)
IL-1120573 17kDa
120573-actin 42kDa
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(b)
Figure 4 (a) Quantitative analysis of mRNA expression by real-time PCR showed a marked increase of IL-1120573 mRNA expression in thebrain of P6 rat pups that were kept for 24 h under hyperoxia (black bar) whereas dexmedetomidine treatment restores IL-1120573 upon controllevel (hatched grey bars) depending on the dexmedetomidine concentration Application of dexmedetomidine under room air (grey bars)showed no significant regulation on IL-1120573 mRNA expression (b) The analysis of IL-1120573 protein expression by western blot showed a similarexpression pattern The protein expression of IL-1120573 is significantly increased after 24 h of hyperoxia and a single application of 5 or 10 120583gkgdexmedetomidine could restore the IL-1120573 protein expression almost up to control level The densitometric data represent the ratio of thepixel intensity of the IL-1120573 band to the corresponding 120573-actin band Blots are representative of a series of three blots Data are normalizedto levels of rat pups exposed to normoxia (CON = 100 white bars) Bars represent mean + SEM 119899 = 6 per group lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 005 119875 lt 001 and
119875 lt 0001 versus hyperoxia
up to normoxia levels in hyperoxia-exposed neonatal ratsthat received dexmedetomidine (Figures 2 and 3) Theseobservations suggest that dexmedetomidine seemingly hasan antioxidant activity against hyperoxia-induced oxidativestress in the developing brain In line with our findings thereare several studies that suggest an increased antioxidativecapacity of dexmedetomidine in the brain [24] and furtherorgans (reviewed by Tse et al [45])
This study indicates that dexmedetomidine significantlydecreases hyperoxia-induced IL-1120573 upregulation in the devel-oping rat brainThese results illustrate the protective effect ofdexmedetomidine on neuroinflammation as shown before inclinical and experimental studies [46 47] Dexmedetomidineitself did not affect IL-1120573 levels indicating that the 1205722-adrenoceptor agonist only acts to normalize the inducedproinflammatory cytokine (Figure 4) Here dexmedetomi-dine appears to inhibit the production of inflammatorymediators from multiple cell types for example astrocytesand microglia [48 49]
There are several limitations of this study pointing to areasof future investigations Dexmedetomidine was given onlyonce to the animals while preterm infants receive an initialloading dose and subsequent infusions [50] and the highdoses of dexmedetomidine studied here exceed those dosesrecommended for clinical use short term hyperoxia wasexamined and preterm infants are often exposed to a longerperiod of supraphysiological oxygen concentrations [51] andwe have not determined the plasma levels of dexmedeto-midine Further details of dexmedetomidine-induced sideeffects remain to be elucidated
To our knowledge this is the first report demonstratingthat dexmedetomidine mediates a decrease of neurodegen-eration and oxidative stress and affects the expression of theproinflammatory cytokine IL-1120573 in the hyperoxia-exposedneonatal brain Since oxidative stress and inflammation areinvolved in dysfunction of the immature brain under highoxygen conditions 1205722-adrenoceptor activation seems to bea potential neuroprotective treatment
Oxidative Medicine and Cellular Longevity 9
5 Conclusion
The essential core of this work is the finding that dexmedeto-midine prevents oxidative stress inflammation and celldeath in the neonatal brain under hyperoxic conditionsExposure to high oxygen in the developing brain of six-day-old rat pups leads to increased oxidative stress-induced andinflammatoryDNAdamage as demonstrated by an increasedapoptotic rate a reduction of the GSHGSSG ratio increasedlipid peroxidation and increased IL-1120573 levels Remarkably asingle dose of dexmedetomidine has weakened or abolishedall these detrimental effects under hyperoxic conditionsHowever in this study dexmedetomidine also shows negativeeffects in the control animals and these potential side effectsshould be taken into consideration in further studies on theprotection of the immature brain with dexmedetomidine
Based on these data the hyperoxic model indicatesdexmedetomidine as a sedative in pediatric anesthesia anda possible promising agent for neuroprotective strategies inpreterm infants
Conflict of Interests
The authors declare that there is no conflict of interestswith any financial organization regarding the commercialidentities mentioned in the paper
Acknowledgments
This work was supported by the Forderverein fur fruh-geborene Kinder an der Charite eV Berlin The authorsgratefully thank Evelyn Strauss and Vanessa Valdix for excel-lent technical assistance
References
[1] E Gerdin O Tyden and U J Eriksson ldquoThe developmentof antioxidant enzymatic defense in the perinatal rat lungactivities of superoxide dismutase glutathione peroxidase andcatalaserdquo Pediatric Research vol 19 no 7 pp 687ndash691 1985
[2] R E Black S Cousens H L Johnson et al ldquoGlobal regionaland national causes of child mortality in 2008 a systematicanalysisrdquoThe Lancet vol 375 no 9730 pp 1969ndash1987 2010
[3] M C Allen ldquoNeurodevelopmental outcomes of preterminfantsrdquo Current Opinion in Neurology vol 21 no 2 pp 123ndash128 2008
[4] B R Vohr ldquoNeurodevelopmental outcomes of extremelypreterm infantsrdquo Clinics in Perinatology vol 41 no 1 pp 241ndash255 2014
[5] K J S Anand and R W Hall ldquoPharmacological therapy foranalgesia and sedation in the newbornrdquo Archives of Disease inChildhood Fetal and Neonatal Edition vol 91 no 6 pp F448ndashF453 2006
[6] CMahajan andHHDash ldquoProcedural sedation and analgesiain pediatric patientsrdquo Journal of Pediatric Neurosciences vol 9no 1 pp 1ndash6 2014
[7] S G Soriano andK J S Anand ldquoAnesthetics and brain toxicityrdquoCurrent Opinion in Anaesthesiology vol 18 no 3 pp 293ndash2972005
[8] C McPherson ldquoSedation and analgesia in mechanically ven-tilated preterm neonates continue standard of care or experi-mentrdquoThe Journal of Pediatric Pharmacology andTherapeuticsvol 17 no 4 pp 351ndash364 2012
[9] R Deulofeut D Golde and S Augusto ldquoTreatment-by-gendereffect when aiming to avoid hyperoxia in preterm infants in theNICUrdquo Acta Paediatrica vol 96 no 7 pp 990ndash994 2007
[10] C J Wright and P A Dennery ldquoManipulation of gene expres-sion by oxygen a primer from bedside to benchrdquo PediatricResearch vol 66 no 1 pp 3ndash10 2009
[11] O D Saugstad M Vento S Ramji D Howard and R FSoll ldquoNeurodevelopmental outcome of infants resuscitatedwithair or 100 oxygen a systematic review and meta-analysisrdquoNeonatology vol 102 no 2 pp 98ndash103 2012
[12] U Felderhoff-Mueser P Bittigau M Sifringer et al ldquoOxygencauses cell death in the developing brainrdquo Neurobiology ofDisease vol 17 no 2 pp 273ndash282 2004
[13] M Sifringer K Genz D Brait et al ldquoErythropoietin attenuateshyperoxia-induced cell death by modulation of inflammatorymediators and matrix metalloproteinasesrdquoDevelopmental Neu-roscience vol 31 no 5 pp 394ndash402 2009
[14] M Sifringer D Brait U Weichelt et al ldquoErythropoietinattenuates hyperoxia-induced oxidative stress in the developingrat brainrdquoBrain Behavior and Immunity vol 24 no 5 pp 792ndash799 2010
[15] M Sifringer I Bendix C von Haefen et al ldquoOxygen toxicityis reduced by acetylcholinesterase inhibition in the developingrat brainrdquoDevelopmental Neuroscience vol 35 no 2-3 pp 255ndash264 2013
[16] U Felderhoff-Mueser M Sifringer O Polley et al ldquoCaspase-1-processed interleukins in hyperoxia-induced cell death in thedeveloping brainrdquo Annals of Neurology vol 57 no 1 pp 50ndash592005
[17] S Endesfelder I Zaak U Weichelt C Buhrer and T SchmitzldquoCaffeine protects neuronal cells against injury caused byhyperoxia in the immature brainrdquo Free Radical Biology andMedicine vol 67 pp 221ndash234 2014
[18] F Brehmer I Bendix S Prager et al ldquoInteraction of inflam-mation and hyperoxia in a rat model of neonatal white matterdamagerdquo PLoS ONE vol 7 no 11 Article ID e49023 2012
[19] J Ard W Doyle and A Bekker ldquoAwake craniotomy withdexmedetomidine in pediatric patientsrdquo Journal of Neurosurgi-cal Anesthesiology vol 15 no 3 pp 263ndash266 2003
[20] M A E Ramsay and D L Luterman ldquoDexmedetomidine as atotal intravenous anesthetic agentrdquo Anesthesiology vol 101 no3 pp 787ndash790 2004
[21] S M Walker R F Howard K A Keay and M FitzgeraldldquoDevelopmental age influences the effect of epidural dexmedet-omidine on inflammatory hyperalgesia in rat pupsrdquo Anesthesi-ology vol 102 no 6 pp 1226ndash1234 2005
[22] J Kuhmonen J Pokorny R Miettinen et al ldquoNeuroprotectiveeffects of dexmedetomidine in the gerbil hippocampus aftertransient global ischemiardquoAnesthesiology vol 87 no 2 pp 371ndash377 1997
[23] D Ma M Hossain N Rajakumaraswamy et al ldquoDexmedeto-midine produces its neuroprotective effect via the alpha 2A-adrenoceptor subtyperdquo European Journal of Pharmacology vol502 no 1-2 pp 87ndash97 2004
[24] O Eser H Fidan O Sahin et al ldquoThe influence of dexmedeto-midine on ischemic rat hippocampusrdquo Brain Research vol 1218pp 250ndash256 2008
10 Oxidative Medicine and Cellular Longevity
[25] M Cosar O Eser H Fidan et al ldquoThe neuroprotective effectof dexmedetomidine in the hippocampus of rabbits after sub-arachnoid hemorrhagerdquo Surgical Neurology vol 71 no 1 pp54ndash59 2009
[26] X Duan Y Li C Zhou L Huang and Z Dong ldquoDexmedeto-midine provides neuroprotection impact on ketamine-inducedneuroapoptosis in the developing rat brainrdquo Acta Anaesthesio-logica Scandinavica vol 58 no 9 pp 1121ndash1126 2014
[27] B Xiong Q-Q Shi and C-H Miao ldquoDexmedetomidinerenders a brain protection on hippocampal formation throughinhibition of nNOS-NO signalling in endotoxin-induced shockratsrdquo Brain Injury vol 28 no 7 pp 1003ndash1008 2014
[28] R M Venn J Hell and R M Grounds ldquoRespiratory effectsof dexmedetomidine in the surgical patient requiring intensivecarerdquo Critical Care vol 4 no 5 pp 302ndash308 2000
[29] J D Tobias and J W Berkenbosch ldquoSedation during mechani-cal ventilation in infants and children dexmedetomidine versusmidazolamrdquo Southern Medical Journal vol 97 no 5 pp 451ndash455 2004
[30] J D Tobias ldquoDexmedetomidine to treat opioid withdrawalin infants following prolonged sedation in the pediatric ICUrdquoJournal of Opioid Management vol 2 no 4 pp 201ndash205 2006
[31] R D Sanders J Xu Y Shu et al ldquoDexmedetomidine attenu-ates isoflurane-induced neurocognitive impairment in neonatalratsrdquo Anesthesiology vol 110 no 5 pp 1077ndash1085 2009
[32] R D Sanders P Sun S Patel M Li M Maze and D MaldquoDexmedetomidine provides cortical neuroprotection impacton anaesthetic-induced neuroapoptosis in the rat developingbrainrdquo Acta Anaesthesiologica Scandinavica vol 54 no 6 pp710ndash716 2010
[33] R R Riker Y Shehabi P M Bokesch et al ldquoDexmedetomidinevsmidazolam for sedation of critically Ill patients a randomizedtrialrdquoThe Journal of the American Medical Association vol 301no 5 pp 489ndash499 2009
[34] S M Jakob E Ruokonen R M Grounds et al ldquoDexmedeto-midine vsmidazolamor propofol for sedation during prolongedmechanical ventilation two randomized controlled trialsrdquo TheJournal of the American Medical Association vol 307 no 11 pp1151ndash1160 2012
[35] K OrsquoMara P Gal J L Ransom et al ldquoSuccessful use of dexme-detomidine for sedation in a 24-week gestational age neonaterdquoThe Annals of Pharmacotherapy vol 43 no 10 pp 1707ndash17132009
[36] K OrsquoMara P Gal J Wimmer et al ldquoDexmedetomidine versusstandard therapy with fentanyl for sedation in mechanicallyventilated premature neonatesrdquo The Journal of Pediatric Phar-macology andTherapeutics vol 17 no 3 pp 252ndash262 2012
[37] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[38] U Yis S H Kurul A Kumral et al ldquoHyperoxic exposure leadsto cell death in the developing brainrdquo Brain ampDevelopment vol30 no 9 pp 556ndash562 2008
[39] K Engelhard C Werner S Kaspar et al ldquoEffect of thealpha2-agonist dexmedetomidine on cerebral neurotransmitterconcentrations during cerebral ischemia in ratsrdquoAnesthesiologyvol 96 no 2 pp 450ndash457 2002
[40] Y Li M Zeng W Chen et al ldquoDexmedetomidine reducesisoflurane-induced neuroapoptosis partly by preserving PI3KAkt pathway in the hippocampus of neonatal ratsrdquo PLoS ONEvol 9 no 4 Article ID e93639 2014
[41] AM KaindlM Sifringer A Koppelstaetter et al ldquoErythropoi-etin protects the developing brain from hyperoxia-induced celldeath and proteome changesrdquo Annals of Neurology vol 64 no5 pp 523ndash534 2008
[42] E Koo T Oshodi CMeschter A Ebrahimnejad and G DongldquoNeurotoxic effects of dexmedetomidine in fetal cynomolgusmonkey brainsrdquo Journal of Toxicological Sciences vol 39 no 2pp 251ndash262 2014
[43] H Ayoglu S Gul V Hanci et al ldquoThe effects of dexmedeto-midine dosage on cerebral vasospasm in a rat subarachnoidhaemorrhage modelrdquo Journal of Clinical Neuroscience vol 17no 6 pp 770ndash773 2010
[44] T Nakano and H Okamoto ldquoDexmedetomidine-induced cere-bral hypoperfusion exacerbates ischemic brain injury in ratsrdquoJournal of Anesthesia vol 23 no 3 pp 378ndash384 2009
[45] I Tse H L Zhao and D Q Ma ldquoOrganoprotective effects ofDexmedetomidine from bench to bedsiderdquo Journal of Periop-erative Science vol 1 no 3 pp 1ndash15 2014
[46] M Tasdogan D Memis N Sut and M Yuksel ldquoResults of apilot study on the effects of propofol and dexmedetomidine oninflammatory responses and intraabdominal pressure in severesepsisrdquo Journal of Clinical Anesthesia vol 21 no 6 pp 394ndash4002009
[47] K Tanabe R Matsushima-Nishiwaki O Kozawa and HIida ldquoDexmedetomidine suppresses interleukin-1120573-inducedinterleukin-6 synthesis in rat glial cellsrdquo International Journalof Molecular Medicine vol 34 no 4 pp 1032ndash1038 2014
[48] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitstumor necrosis factor-alpha and interleukin 6 in lipopoly-saccharide-stimulated astrocytes by suppression of c-Jun N-terminal kinasesrdquo Inflammation vol 37 no 3 pp 942ndash9492014
[49] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitsinducible nitric oxide synthase in lipopolysaccharide-stimulat-ed microglia by suppression of extracellular signal-regulatedkinaserdquo Neurological Research vol 37 no 3 pp 238ndash245 2015
[50] C Chrysostomou S R Schulman M H Castellanos et al ldquoAphase IIIII multicenter safety efficacy and pharmacokineticstudy of dexmedetomidine in preterm and term neonatesrdquoTheJournal of Pediatrics vol 164 no 2 pp 276e3ndash282e3 2014
[51] K Konig and K J Guy ldquoBronchopulmonary dysplasia inpreterm infants managed with non-invasive ventilation orsurfactant and a brief period of mechanical ventilation a 6-year cohort studyrdquo The Journal of Maternal-Fetal amp NeonatalMedicine vol 27 no 6 pp 608ndash611 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 7
Lipid peroxidation
00
25
50
75
100
125
150
175
MD
A (n
mol
mg
prot
ein)
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
lowastlowastlowast
lowastlowastlowastlowastlowastlowastlowastlowastlowast
lowast
Figure 3 Alteration of lipid peroxidation by hyperoxia in the imma-ture brain Hyperoxia lead to a significant increase of MDA levelsafter 24 h of oxygen exposure (black bar) whereas a single DEXapplication of 5 or 10 120583gkg (hatched grey bars) before hyperoxiaexposure reduced these levels significantly Bars represent mean +SEM 119899 = 6 per group lowast119875 lt 005 and lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 001 and
119875 lt 0001 versus hyperoxia
animals showed low mRNA levels of IL-1120573 (CON 10036 +1141) When dexmedetomidine was administered togetherwith hyperoxia it significantly ameliorated the expression ofproinflammatory IL-1120573 depending on theDEX concentration(HYDEX5 31029 + 2177 and HYDEX10 17766 + 2233)on mRNA levels in rat pups Pretreatment with DEX inanimals kept under normoxic conditions had no effect onIL-1120573 gene expression (DEX1 11718 + 1158 DEX5 12141 +914 and DEX10 13531 + 1285)
Western blotting demonstrated that protein expression ofIL-1120573 is significantly increased following oxygen treatmentfor 24 h (Figure 4(b) HY 33666 + 1725) compared tocontrol animals (CON 10000 + 731) A significant effect onIL-1120573 protein levels was seen upon a single dexmedetomidineapplication of 5 or 10 120583gkg DEX in hyperoxia-exposedanimals (HYDEX5 25096 + 1355 and HYDEX10 17105+ 1227) Animals under normoxic conditions and DEXpretreatment showed low protein levels of IL-1120573 (DEX1 11195+ 443 DEX5 10786 + 515 and DEX10 11244 + 543)
4 Discussion
In the present study we demonstrate that the 1205722-adren-oceptor agonist dexmedetomidine leads to a decrease ofneurodegeneration and affects the level of oxidative stressparameters and the proinflammatory cytokine IL-1120573 in amodel of neonatal hyperoxia-induced brain injury
As shown previously oxygen toxicity leads to an increasedneurodegeneration in the immature brain [12 16 17 38] Inline with these studies here we indicate that an exposure ofsix-day-old rats to a high oxygen concentration (FiO
280)
over 24 h resulted in an increase of neurodegeneration to theneonatal brain (Figures 1(a) and 1(b))Moreover we point outa significant decrease of cell death in the developing rat brainwhen infant rats were treated intraperitoneally with 1 5 or10 120583gkg dexmedetomidine before exposure to high oxygenconditions These dosages are atunder the lower levels ofdexmedetomidine concentrations used in common adult ratmodels of neuronal damage [39 40] and the present dataare not intended to demonstrate the evidence of an effectivedose However our findings conform with different otheranimal studies which examined the neuroprotective capac-ity of dexmedetomidine [22ndash27] Hyperoxia-exposed ratsrevealed an up to 5-fold increase in cell degeneration withinthe cortex and deep gray matter (Figure 1(b)) which hasbeen demonstrated in previous studies [17 41] Interestinglydexmedetomidine treatment showed a profound decrease ofdegenerating cells in all analysed brain regions of rats exposedto hyperoxia (Figure 1(b)) Dexmedetomidine alone inducedno oxidative stress but at a relatively low concentration ofdexmedetomidine under normoxic conditions a significantincrease in apoptosis in the cortex and the thalamus hasbeen shown This result is controversial to other inventions[31 32 42] and to the description of only protective andno toxic effects [24 25 43] In vitro it was shown byKuhmonen et al [22] that dexmedetomidine is more effectiveat lower doses and protects against delayed cell death In anischemia-reperfusionmodel [39] antiapoptotic proteins wereupregulated and proapoptotic proteins are suppressed bydexmedetomidine treatment In the study by Li et al there areno changes in the expression of proapoptotic mediators in anisoflurane-induced model of neuroapoptosis [40] A provenhigh blood pressure after the administration of high-dosedexmedetomidine correlates with cerebral hypoperfusionmay be due to alpha-2-induced cerebral vasoconstriction[44] so that this could contribute to apoptotic cell deathtriggered by other cellular mechanisms However in ourstudy dexmedetomidine also led to negative effects in controlanimals notably in the thalamus and potential side effectsshould be further considered in studies on protection of theimmature brain using dexmedetomidine
The potency of dexmedetomidine in this model of oxy-gen toxicity indicates that the 1205722-adrenoceptor agonist hasantioxidant activities Of note our hyperoxia model is pre-dominantly based on free radical generation as we previouslyfound an induction of different oxidative stress parameters[12ndash15] Therefore we evaluated the effects on biochemicalmarkers of oxidative stress in the developing brain Theconcentrations of GSH and GSSG the GSHGSSG ratio andthe MDA level were used as indicators of oxidative stressand lipid breakdown Hyperoxia-exposed P6 rats showedsignificant decreased GSH and increased GSSG levels at24 h of hyperoxia In addition a decrease in GSHGSSGratio and an increase in MDA level in the developingbrain indicate neuronal damage due to oxidative stressbut there was a modulation of the levels of all parameters
8 Oxidative Medicine and Cellular Longevity
0
100
200
300
400
500
600
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowast
IL-1120573 gene expression
Rela
tive I
L-1120573
gene
expr
essio
n2minusΔΔ
Ct(
)
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(a)
0
100
200
300
400
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
IL-1120573 protein expression
Den
sity
ratio
IL-1120573
120573-a
ctin
( o
f con
trol)
IL-1120573 17kDa
120573-actin 42kDa
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(b)
Figure 4 (a) Quantitative analysis of mRNA expression by real-time PCR showed a marked increase of IL-1120573 mRNA expression in thebrain of P6 rat pups that were kept for 24 h under hyperoxia (black bar) whereas dexmedetomidine treatment restores IL-1120573 upon controllevel (hatched grey bars) depending on the dexmedetomidine concentration Application of dexmedetomidine under room air (grey bars)showed no significant regulation on IL-1120573 mRNA expression (b) The analysis of IL-1120573 protein expression by western blot showed a similarexpression pattern The protein expression of IL-1120573 is significantly increased after 24 h of hyperoxia and a single application of 5 or 10 120583gkgdexmedetomidine could restore the IL-1120573 protein expression almost up to control level The densitometric data represent the ratio of thepixel intensity of the IL-1120573 band to the corresponding 120573-actin band Blots are representative of a series of three blots Data are normalizedto levels of rat pups exposed to normoxia (CON = 100 white bars) Bars represent mean + SEM 119899 = 6 per group lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 005 119875 lt 001 and
119875 lt 0001 versus hyperoxia
up to normoxia levels in hyperoxia-exposed neonatal ratsthat received dexmedetomidine (Figures 2 and 3) Theseobservations suggest that dexmedetomidine seemingly hasan antioxidant activity against hyperoxia-induced oxidativestress in the developing brain In line with our findings thereare several studies that suggest an increased antioxidativecapacity of dexmedetomidine in the brain [24] and furtherorgans (reviewed by Tse et al [45])
This study indicates that dexmedetomidine significantlydecreases hyperoxia-induced IL-1120573 upregulation in the devel-oping rat brainThese results illustrate the protective effect ofdexmedetomidine on neuroinflammation as shown before inclinical and experimental studies [46 47] Dexmedetomidineitself did not affect IL-1120573 levels indicating that the 1205722-adrenoceptor agonist only acts to normalize the inducedproinflammatory cytokine (Figure 4) Here dexmedetomi-dine appears to inhibit the production of inflammatorymediators from multiple cell types for example astrocytesand microglia [48 49]
There are several limitations of this study pointing to areasof future investigations Dexmedetomidine was given onlyonce to the animals while preterm infants receive an initialloading dose and subsequent infusions [50] and the highdoses of dexmedetomidine studied here exceed those dosesrecommended for clinical use short term hyperoxia wasexamined and preterm infants are often exposed to a longerperiod of supraphysiological oxygen concentrations [51] andwe have not determined the plasma levels of dexmedeto-midine Further details of dexmedetomidine-induced sideeffects remain to be elucidated
To our knowledge this is the first report demonstratingthat dexmedetomidine mediates a decrease of neurodegen-eration and oxidative stress and affects the expression of theproinflammatory cytokine IL-1120573 in the hyperoxia-exposedneonatal brain Since oxidative stress and inflammation areinvolved in dysfunction of the immature brain under highoxygen conditions 1205722-adrenoceptor activation seems to bea potential neuroprotective treatment
Oxidative Medicine and Cellular Longevity 9
5 Conclusion
The essential core of this work is the finding that dexmedeto-midine prevents oxidative stress inflammation and celldeath in the neonatal brain under hyperoxic conditionsExposure to high oxygen in the developing brain of six-day-old rat pups leads to increased oxidative stress-induced andinflammatoryDNAdamage as demonstrated by an increasedapoptotic rate a reduction of the GSHGSSG ratio increasedlipid peroxidation and increased IL-1120573 levels Remarkably asingle dose of dexmedetomidine has weakened or abolishedall these detrimental effects under hyperoxic conditionsHowever in this study dexmedetomidine also shows negativeeffects in the control animals and these potential side effectsshould be taken into consideration in further studies on theprotection of the immature brain with dexmedetomidine
Based on these data the hyperoxic model indicatesdexmedetomidine as a sedative in pediatric anesthesia anda possible promising agent for neuroprotective strategies inpreterm infants
Conflict of Interests
The authors declare that there is no conflict of interestswith any financial organization regarding the commercialidentities mentioned in the paper
Acknowledgments
This work was supported by the Forderverein fur fruh-geborene Kinder an der Charite eV Berlin The authorsgratefully thank Evelyn Strauss and Vanessa Valdix for excel-lent technical assistance
References
[1] E Gerdin O Tyden and U J Eriksson ldquoThe developmentof antioxidant enzymatic defense in the perinatal rat lungactivities of superoxide dismutase glutathione peroxidase andcatalaserdquo Pediatric Research vol 19 no 7 pp 687ndash691 1985
[2] R E Black S Cousens H L Johnson et al ldquoGlobal regionaland national causes of child mortality in 2008 a systematicanalysisrdquoThe Lancet vol 375 no 9730 pp 1969ndash1987 2010
[3] M C Allen ldquoNeurodevelopmental outcomes of preterminfantsrdquo Current Opinion in Neurology vol 21 no 2 pp 123ndash128 2008
[4] B R Vohr ldquoNeurodevelopmental outcomes of extremelypreterm infantsrdquo Clinics in Perinatology vol 41 no 1 pp 241ndash255 2014
[5] K J S Anand and R W Hall ldquoPharmacological therapy foranalgesia and sedation in the newbornrdquo Archives of Disease inChildhood Fetal and Neonatal Edition vol 91 no 6 pp F448ndashF453 2006
[6] CMahajan andHHDash ldquoProcedural sedation and analgesiain pediatric patientsrdquo Journal of Pediatric Neurosciences vol 9no 1 pp 1ndash6 2014
[7] S G Soriano andK J S Anand ldquoAnesthetics and brain toxicityrdquoCurrent Opinion in Anaesthesiology vol 18 no 3 pp 293ndash2972005
[8] C McPherson ldquoSedation and analgesia in mechanically ven-tilated preterm neonates continue standard of care or experi-mentrdquoThe Journal of Pediatric Pharmacology andTherapeuticsvol 17 no 4 pp 351ndash364 2012
[9] R Deulofeut D Golde and S Augusto ldquoTreatment-by-gendereffect when aiming to avoid hyperoxia in preterm infants in theNICUrdquo Acta Paediatrica vol 96 no 7 pp 990ndash994 2007
[10] C J Wright and P A Dennery ldquoManipulation of gene expres-sion by oxygen a primer from bedside to benchrdquo PediatricResearch vol 66 no 1 pp 3ndash10 2009
[11] O D Saugstad M Vento S Ramji D Howard and R FSoll ldquoNeurodevelopmental outcome of infants resuscitatedwithair or 100 oxygen a systematic review and meta-analysisrdquoNeonatology vol 102 no 2 pp 98ndash103 2012
[12] U Felderhoff-Mueser P Bittigau M Sifringer et al ldquoOxygencauses cell death in the developing brainrdquo Neurobiology ofDisease vol 17 no 2 pp 273ndash282 2004
[13] M Sifringer K Genz D Brait et al ldquoErythropoietin attenuateshyperoxia-induced cell death by modulation of inflammatorymediators and matrix metalloproteinasesrdquoDevelopmental Neu-roscience vol 31 no 5 pp 394ndash402 2009
[14] M Sifringer D Brait U Weichelt et al ldquoErythropoietinattenuates hyperoxia-induced oxidative stress in the developingrat brainrdquoBrain Behavior and Immunity vol 24 no 5 pp 792ndash799 2010
[15] M Sifringer I Bendix C von Haefen et al ldquoOxygen toxicityis reduced by acetylcholinesterase inhibition in the developingrat brainrdquoDevelopmental Neuroscience vol 35 no 2-3 pp 255ndash264 2013
[16] U Felderhoff-Mueser M Sifringer O Polley et al ldquoCaspase-1-processed interleukins in hyperoxia-induced cell death in thedeveloping brainrdquo Annals of Neurology vol 57 no 1 pp 50ndash592005
[17] S Endesfelder I Zaak U Weichelt C Buhrer and T SchmitzldquoCaffeine protects neuronal cells against injury caused byhyperoxia in the immature brainrdquo Free Radical Biology andMedicine vol 67 pp 221ndash234 2014
[18] F Brehmer I Bendix S Prager et al ldquoInteraction of inflam-mation and hyperoxia in a rat model of neonatal white matterdamagerdquo PLoS ONE vol 7 no 11 Article ID e49023 2012
[19] J Ard W Doyle and A Bekker ldquoAwake craniotomy withdexmedetomidine in pediatric patientsrdquo Journal of Neurosurgi-cal Anesthesiology vol 15 no 3 pp 263ndash266 2003
[20] M A E Ramsay and D L Luterman ldquoDexmedetomidine as atotal intravenous anesthetic agentrdquo Anesthesiology vol 101 no3 pp 787ndash790 2004
[21] S M Walker R F Howard K A Keay and M FitzgeraldldquoDevelopmental age influences the effect of epidural dexmedet-omidine on inflammatory hyperalgesia in rat pupsrdquo Anesthesi-ology vol 102 no 6 pp 1226ndash1234 2005
[22] J Kuhmonen J Pokorny R Miettinen et al ldquoNeuroprotectiveeffects of dexmedetomidine in the gerbil hippocampus aftertransient global ischemiardquoAnesthesiology vol 87 no 2 pp 371ndash377 1997
[23] D Ma M Hossain N Rajakumaraswamy et al ldquoDexmedeto-midine produces its neuroprotective effect via the alpha 2A-adrenoceptor subtyperdquo European Journal of Pharmacology vol502 no 1-2 pp 87ndash97 2004
[24] O Eser H Fidan O Sahin et al ldquoThe influence of dexmedeto-midine on ischemic rat hippocampusrdquo Brain Research vol 1218pp 250ndash256 2008
10 Oxidative Medicine and Cellular Longevity
[25] M Cosar O Eser H Fidan et al ldquoThe neuroprotective effectof dexmedetomidine in the hippocampus of rabbits after sub-arachnoid hemorrhagerdquo Surgical Neurology vol 71 no 1 pp54ndash59 2009
[26] X Duan Y Li C Zhou L Huang and Z Dong ldquoDexmedeto-midine provides neuroprotection impact on ketamine-inducedneuroapoptosis in the developing rat brainrdquo Acta Anaesthesio-logica Scandinavica vol 58 no 9 pp 1121ndash1126 2014
[27] B Xiong Q-Q Shi and C-H Miao ldquoDexmedetomidinerenders a brain protection on hippocampal formation throughinhibition of nNOS-NO signalling in endotoxin-induced shockratsrdquo Brain Injury vol 28 no 7 pp 1003ndash1008 2014
[28] R M Venn J Hell and R M Grounds ldquoRespiratory effectsof dexmedetomidine in the surgical patient requiring intensivecarerdquo Critical Care vol 4 no 5 pp 302ndash308 2000
[29] J D Tobias and J W Berkenbosch ldquoSedation during mechani-cal ventilation in infants and children dexmedetomidine versusmidazolamrdquo Southern Medical Journal vol 97 no 5 pp 451ndash455 2004
[30] J D Tobias ldquoDexmedetomidine to treat opioid withdrawalin infants following prolonged sedation in the pediatric ICUrdquoJournal of Opioid Management vol 2 no 4 pp 201ndash205 2006
[31] R D Sanders J Xu Y Shu et al ldquoDexmedetomidine attenu-ates isoflurane-induced neurocognitive impairment in neonatalratsrdquo Anesthesiology vol 110 no 5 pp 1077ndash1085 2009
[32] R D Sanders P Sun S Patel M Li M Maze and D MaldquoDexmedetomidine provides cortical neuroprotection impacton anaesthetic-induced neuroapoptosis in the rat developingbrainrdquo Acta Anaesthesiologica Scandinavica vol 54 no 6 pp710ndash716 2010
[33] R R Riker Y Shehabi P M Bokesch et al ldquoDexmedetomidinevsmidazolam for sedation of critically Ill patients a randomizedtrialrdquoThe Journal of the American Medical Association vol 301no 5 pp 489ndash499 2009
[34] S M Jakob E Ruokonen R M Grounds et al ldquoDexmedeto-midine vsmidazolamor propofol for sedation during prolongedmechanical ventilation two randomized controlled trialsrdquo TheJournal of the American Medical Association vol 307 no 11 pp1151ndash1160 2012
[35] K OrsquoMara P Gal J L Ransom et al ldquoSuccessful use of dexme-detomidine for sedation in a 24-week gestational age neonaterdquoThe Annals of Pharmacotherapy vol 43 no 10 pp 1707ndash17132009
[36] K OrsquoMara P Gal J Wimmer et al ldquoDexmedetomidine versusstandard therapy with fentanyl for sedation in mechanicallyventilated premature neonatesrdquo The Journal of Pediatric Phar-macology andTherapeutics vol 17 no 3 pp 252ndash262 2012
[37] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[38] U Yis S H Kurul A Kumral et al ldquoHyperoxic exposure leadsto cell death in the developing brainrdquo Brain ampDevelopment vol30 no 9 pp 556ndash562 2008
[39] K Engelhard C Werner S Kaspar et al ldquoEffect of thealpha2-agonist dexmedetomidine on cerebral neurotransmitterconcentrations during cerebral ischemia in ratsrdquoAnesthesiologyvol 96 no 2 pp 450ndash457 2002
[40] Y Li M Zeng W Chen et al ldquoDexmedetomidine reducesisoflurane-induced neuroapoptosis partly by preserving PI3KAkt pathway in the hippocampus of neonatal ratsrdquo PLoS ONEvol 9 no 4 Article ID e93639 2014
[41] AM KaindlM Sifringer A Koppelstaetter et al ldquoErythropoi-etin protects the developing brain from hyperoxia-induced celldeath and proteome changesrdquo Annals of Neurology vol 64 no5 pp 523ndash534 2008
[42] E Koo T Oshodi CMeschter A Ebrahimnejad and G DongldquoNeurotoxic effects of dexmedetomidine in fetal cynomolgusmonkey brainsrdquo Journal of Toxicological Sciences vol 39 no 2pp 251ndash262 2014
[43] H Ayoglu S Gul V Hanci et al ldquoThe effects of dexmedeto-midine dosage on cerebral vasospasm in a rat subarachnoidhaemorrhage modelrdquo Journal of Clinical Neuroscience vol 17no 6 pp 770ndash773 2010
[44] T Nakano and H Okamoto ldquoDexmedetomidine-induced cere-bral hypoperfusion exacerbates ischemic brain injury in ratsrdquoJournal of Anesthesia vol 23 no 3 pp 378ndash384 2009
[45] I Tse H L Zhao and D Q Ma ldquoOrganoprotective effects ofDexmedetomidine from bench to bedsiderdquo Journal of Periop-erative Science vol 1 no 3 pp 1ndash15 2014
[46] M Tasdogan D Memis N Sut and M Yuksel ldquoResults of apilot study on the effects of propofol and dexmedetomidine oninflammatory responses and intraabdominal pressure in severesepsisrdquo Journal of Clinical Anesthesia vol 21 no 6 pp 394ndash4002009
[47] K Tanabe R Matsushima-Nishiwaki O Kozawa and HIida ldquoDexmedetomidine suppresses interleukin-1120573-inducedinterleukin-6 synthesis in rat glial cellsrdquo International Journalof Molecular Medicine vol 34 no 4 pp 1032ndash1038 2014
[48] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitstumor necrosis factor-alpha and interleukin 6 in lipopoly-saccharide-stimulated astrocytes by suppression of c-Jun N-terminal kinasesrdquo Inflammation vol 37 no 3 pp 942ndash9492014
[49] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitsinducible nitric oxide synthase in lipopolysaccharide-stimulat-ed microglia by suppression of extracellular signal-regulatedkinaserdquo Neurological Research vol 37 no 3 pp 238ndash245 2015
[50] C Chrysostomou S R Schulman M H Castellanos et al ldquoAphase IIIII multicenter safety efficacy and pharmacokineticstudy of dexmedetomidine in preterm and term neonatesrdquoTheJournal of Pediatrics vol 164 no 2 pp 276e3ndash282e3 2014
[51] K Konig and K J Guy ldquoBronchopulmonary dysplasia inpreterm infants managed with non-invasive ventilation orsurfactant and a brief period of mechanical ventilation a 6-year cohort studyrdquo The Journal of Maternal-Fetal amp NeonatalMedicine vol 27 no 6 pp 608ndash611 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
8 Oxidative Medicine and Cellular Longevity
0
100
200
300
400
500
600
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowast
IL-1120573 gene expression
Rela
tive I
L-1120573
gene
expr
essio
n2minusΔΔ
Ct(
)
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(a)
0
100
200
300
400
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
lowastlowastlowast
IL-1120573 protein expression
Den
sity
ratio
IL-1120573
120573-a
ctin
( o
f con
trol)
IL-1120573 17kDa
120573-actin 42kDa
Hyperoxia
DEX1
DEX5
DEX10
+ +
+
+
+
+
+
+
+
+
minus
minus
minus
minus
minus minus
minus
minus
minus
minus minus minus
minus
minus
minus minus
minus
minusminus
minus
minus
minus
(b)
Figure 4 (a) Quantitative analysis of mRNA expression by real-time PCR showed a marked increase of IL-1120573 mRNA expression in thebrain of P6 rat pups that were kept for 24 h under hyperoxia (black bar) whereas dexmedetomidine treatment restores IL-1120573 upon controllevel (hatched grey bars) depending on the dexmedetomidine concentration Application of dexmedetomidine under room air (grey bars)showed no significant regulation on IL-1120573 mRNA expression (b) The analysis of IL-1120573 protein expression by western blot showed a similarexpression pattern The protein expression of IL-1120573 is significantly increased after 24 h of hyperoxia and a single application of 5 or 10 120583gkgdexmedetomidine could restore the IL-1120573 protein expression almost up to control level The densitometric data represent the ratio of thepixel intensity of the IL-1120573 band to the corresponding 120573-actin band Blots are representative of a series of three blots Data are normalizedto levels of rat pups exposed to normoxia (CON = 100 white bars) Bars represent mean + SEM 119899 = 6 per group lowastlowastlowast119875 lt 0001 versusnormoxiacontrol 119875 lt 005 119875 lt 001 and
119875 lt 0001 versus hyperoxia
up to normoxia levels in hyperoxia-exposed neonatal ratsthat received dexmedetomidine (Figures 2 and 3) Theseobservations suggest that dexmedetomidine seemingly hasan antioxidant activity against hyperoxia-induced oxidativestress in the developing brain In line with our findings thereare several studies that suggest an increased antioxidativecapacity of dexmedetomidine in the brain [24] and furtherorgans (reviewed by Tse et al [45])
This study indicates that dexmedetomidine significantlydecreases hyperoxia-induced IL-1120573 upregulation in the devel-oping rat brainThese results illustrate the protective effect ofdexmedetomidine on neuroinflammation as shown before inclinical and experimental studies [46 47] Dexmedetomidineitself did not affect IL-1120573 levels indicating that the 1205722-adrenoceptor agonist only acts to normalize the inducedproinflammatory cytokine (Figure 4) Here dexmedetomi-dine appears to inhibit the production of inflammatorymediators from multiple cell types for example astrocytesand microglia [48 49]
There are several limitations of this study pointing to areasof future investigations Dexmedetomidine was given onlyonce to the animals while preterm infants receive an initialloading dose and subsequent infusions [50] and the highdoses of dexmedetomidine studied here exceed those dosesrecommended for clinical use short term hyperoxia wasexamined and preterm infants are often exposed to a longerperiod of supraphysiological oxygen concentrations [51] andwe have not determined the plasma levels of dexmedeto-midine Further details of dexmedetomidine-induced sideeffects remain to be elucidated
To our knowledge this is the first report demonstratingthat dexmedetomidine mediates a decrease of neurodegen-eration and oxidative stress and affects the expression of theproinflammatory cytokine IL-1120573 in the hyperoxia-exposedneonatal brain Since oxidative stress and inflammation areinvolved in dysfunction of the immature brain under highoxygen conditions 1205722-adrenoceptor activation seems to bea potential neuroprotective treatment
Oxidative Medicine and Cellular Longevity 9
5 Conclusion
The essential core of this work is the finding that dexmedeto-midine prevents oxidative stress inflammation and celldeath in the neonatal brain under hyperoxic conditionsExposure to high oxygen in the developing brain of six-day-old rat pups leads to increased oxidative stress-induced andinflammatoryDNAdamage as demonstrated by an increasedapoptotic rate a reduction of the GSHGSSG ratio increasedlipid peroxidation and increased IL-1120573 levels Remarkably asingle dose of dexmedetomidine has weakened or abolishedall these detrimental effects under hyperoxic conditionsHowever in this study dexmedetomidine also shows negativeeffects in the control animals and these potential side effectsshould be taken into consideration in further studies on theprotection of the immature brain with dexmedetomidine
Based on these data the hyperoxic model indicatesdexmedetomidine as a sedative in pediatric anesthesia anda possible promising agent for neuroprotective strategies inpreterm infants
Conflict of Interests
The authors declare that there is no conflict of interestswith any financial organization regarding the commercialidentities mentioned in the paper
Acknowledgments
This work was supported by the Forderverein fur fruh-geborene Kinder an der Charite eV Berlin The authorsgratefully thank Evelyn Strauss and Vanessa Valdix for excel-lent technical assistance
References
[1] E Gerdin O Tyden and U J Eriksson ldquoThe developmentof antioxidant enzymatic defense in the perinatal rat lungactivities of superoxide dismutase glutathione peroxidase andcatalaserdquo Pediatric Research vol 19 no 7 pp 687ndash691 1985
[2] R E Black S Cousens H L Johnson et al ldquoGlobal regionaland national causes of child mortality in 2008 a systematicanalysisrdquoThe Lancet vol 375 no 9730 pp 1969ndash1987 2010
[3] M C Allen ldquoNeurodevelopmental outcomes of preterminfantsrdquo Current Opinion in Neurology vol 21 no 2 pp 123ndash128 2008
[4] B R Vohr ldquoNeurodevelopmental outcomes of extremelypreterm infantsrdquo Clinics in Perinatology vol 41 no 1 pp 241ndash255 2014
[5] K J S Anand and R W Hall ldquoPharmacological therapy foranalgesia and sedation in the newbornrdquo Archives of Disease inChildhood Fetal and Neonatal Edition vol 91 no 6 pp F448ndashF453 2006
[6] CMahajan andHHDash ldquoProcedural sedation and analgesiain pediatric patientsrdquo Journal of Pediatric Neurosciences vol 9no 1 pp 1ndash6 2014
[7] S G Soriano andK J S Anand ldquoAnesthetics and brain toxicityrdquoCurrent Opinion in Anaesthesiology vol 18 no 3 pp 293ndash2972005
[8] C McPherson ldquoSedation and analgesia in mechanically ven-tilated preterm neonates continue standard of care or experi-mentrdquoThe Journal of Pediatric Pharmacology andTherapeuticsvol 17 no 4 pp 351ndash364 2012
[9] R Deulofeut D Golde and S Augusto ldquoTreatment-by-gendereffect when aiming to avoid hyperoxia in preterm infants in theNICUrdquo Acta Paediatrica vol 96 no 7 pp 990ndash994 2007
[10] C J Wright and P A Dennery ldquoManipulation of gene expres-sion by oxygen a primer from bedside to benchrdquo PediatricResearch vol 66 no 1 pp 3ndash10 2009
[11] O D Saugstad M Vento S Ramji D Howard and R FSoll ldquoNeurodevelopmental outcome of infants resuscitatedwithair or 100 oxygen a systematic review and meta-analysisrdquoNeonatology vol 102 no 2 pp 98ndash103 2012
[12] U Felderhoff-Mueser P Bittigau M Sifringer et al ldquoOxygencauses cell death in the developing brainrdquo Neurobiology ofDisease vol 17 no 2 pp 273ndash282 2004
[13] M Sifringer K Genz D Brait et al ldquoErythropoietin attenuateshyperoxia-induced cell death by modulation of inflammatorymediators and matrix metalloproteinasesrdquoDevelopmental Neu-roscience vol 31 no 5 pp 394ndash402 2009
[14] M Sifringer D Brait U Weichelt et al ldquoErythropoietinattenuates hyperoxia-induced oxidative stress in the developingrat brainrdquoBrain Behavior and Immunity vol 24 no 5 pp 792ndash799 2010
[15] M Sifringer I Bendix C von Haefen et al ldquoOxygen toxicityis reduced by acetylcholinesterase inhibition in the developingrat brainrdquoDevelopmental Neuroscience vol 35 no 2-3 pp 255ndash264 2013
[16] U Felderhoff-Mueser M Sifringer O Polley et al ldquoCaspase-1-processed interleukins in hyperoxia-induced cell death in thedeveloping brainrdquo Annals of Neurology vol 57 no 1 pp 50ndash592005
[17] S Endesfelder I Zaak U Weichelt C Buhrer and T SchmitzldquoCaffeine protects neuronal cells against injury caused byhyperoxia in the immature brainrdquo Free Radical Biology andMedicine vol 67 pp 221ndash234 2014
[18] F Brehmer I Bendix S Prager et al ldquoInteraction of inflam-mation and hyperoxia in a rat model of neonatal white matterdamagerdquo PLoS ONE vol 7 no 11 Article ID e49023 2012
[19] J Ard W Doyle and A Bekker ldquoAwake craniotomy withdexmedetomidine in pediatric patientsrdquo Journal of Neurosurgi-cal Anesthesiology vol 15 no 3 pp 263ndash266 2003
[20] M A E Ramsay and D L Luterman ldquoDexmedetomidine as atotal intravenous anesthetic agentrdquo Anesthesiology vol 101 no3 pp 787ndash790 2004
[21] S M Walker R F Howard K A Keay and M FitzgeraldldquoDevelopmental age influences the effect of epidural dexmedet-omidine on inflammatory hyperalgesia in rat pupsrdquo Anesthesi-ology vol 102 no 6 pp 1226ndash1234 2005
[22] J Kuhmonen J Pokorny R Miettinen et al ldquoNeuroprotectiveeffects of dexmedetomidine in the gerbil hippocampus aftertransient global ischemiardquoAnesthesiology vol 87 no 2 pp 371ndash377 1997
[23] D Ma M Hossain N Rajakumaraswamy et al ldquoDexmedeto-midine produces its neuroprotective effect via the alpha 2A-adrenoceptor subtyperdquo European Journal of Pharmacology vol502 no 1-2 pp 87ndash97 2004
[24] O Eser H Fidan O Sahin et al ldquoThe influence of dexmedeto-midine on ischemic rat hippocampusrdquo Brain Research vol 1218pp 250ndash256 2008
10 Oxidative Medicine and Cellular Longevity
[25] M Cosar O Eser H Fidan et al ldquoThe neuroprotective effectof dexmedetomidine in the hippocampus of rabbits after sub-arachnoid hemorrhagerdquo Surgical Neurology vol 71 no 1 pp54ndash59 2009
[26] X Duan Y Li C Zhou L Huang and Z Dong ldquoDexmedeto-midine provides neuroprotection impact on ketamine-inducedneuroapoptosis in the developing rat brainrdquo Acta Anaesthesio-logica Scandinavica vol 58 no 9 pp 1121ndash1126 2014
[27] B Xiong Q-Q Shi and C-H Miao ldquoDexmedetomidinerenders a brain protection on hippocampal formation throughinhibition of nNOS-NO signalling in endotoxin-induced shockratsrdquo Brain Injury vol 28 no 7 pp 1003ndash1008 2014
[28] R M Venn J Hell and R M Grounds ldquoRespiratory effectsof dexmedetomidine in the surgical patient requiring intensivecarerdquo Critical Care vol 4 no 5 pp 302ndash308 2000
[29] J D Tobias and J W Berkenbosch ldquoSedation during mechani-cal ventilation in infants and children dexmedetomidine versusmidazolamrdquo Southern Medical Journal vol 97 no 5 pp 451ndash455 2004
[30] J D Tobias ldquoDexmedetomidine to treat opioid withdrawalin infants following prolonged sedation in the pediatric ICUrdquoJournal of Opioid Management vol 2 no 4 pp 201ndash205 2006
[31] R D Sanders J Xu Y Shu et al ldquoDexmedetomidine attenu-ates isoflurane-induced neurocognitive impairment in neonatalratsrdquo Anesthesiology vol 110 no 5 pp 1077ndash1085 2009
[32] R D Sanders P Sun S Patel M Li M Maze and D MaldquoDexmedetomidine provides cortical neuroprotection impacton anaesthetic-induced neuroapoptosis in the rat developingbrainrdquo Acta Anaesthesiologica Scandinavica vol 54 no 6 pp710ndash716 2010
[33] R R Riker Y Shehabi P M Bokesch et al ldquoDexmedetomidinevsmidazolam for sedation of critically Ill patients a randomizedtrialrdquoThe Journal of the American Medical Association vol 301no 5 pp 489ndash499 2009
[34] S M Jakob E Ruokonen R M Grounds et al ldquoDexmedeto-midine vsmidazolamor propofol for sedation during prolongedmechanical ventilation two randomized controlled trialsrdquo TheJournal of the American Medical Association vol 307 no 11 pp1151ndash1160 2012
[35] K OrsquoMara P Gal J L Ransom et al ldquoSuccessful use of dexme-detomidine for sedation in a 24-week gestational age neonaterdquoThe Annals of Pharmacotherapy vol 43 no 10 pp 1707ndash17132009
[36] K OrsquoMara P Gal J Wimmer et al ldquoDexmedetomidine versusstandard therapy with fentanyl for sedation in mechanicallyventilated premature neonatesrdquo The Journal of Pediatric Phar-macology andTherapeutics vol 17 no 3 pp 252ndash262 2012
[37] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[38] U Yis S H Kurul A Kumral et al ldquoHyperoxic exposure leadsto cell death in the developing brainrdquo Brain ampDevelopment vol30 no 9 pp 556ndash562 2008
[39] K Engelhard C Werner S Kaspar et al ldquoEffect of thealpha2-agonist dexmedetomidine on cerebral neurotransmitterconcentrations during cerebral ischemia in ratsrdquoAnesthesiologyvol 96 no 2 pp 450ndash457 2002
[40] Y Li M Zeng W Chen et al ldquoDexmedetomidine reducesisoflurane-induced neuroapoptosis partly by preserving PI3KAkt pathway in the hippocampus of neonatal ratsrdquo PLoS ONEvol 9 no 4 Article ID e93639 2014
[41] AM KaindlM Sifringer A Koppelstaetter et al ldquoErythropoi-etin protects the developing brain from hyperoxia-induced celldeath and proteome changesrdquo Annals of Neurology vol 64 no5 pp 523ndash534 2008
[42] E Koo T Oshodi CMeschter A Ebrahimnejad and G DongldquoNeurotoxic effects of dexmedetomidine in fetal cynomolgusmonkey brainsrdquo Journal of Toxicological Sciences vol 39 no 2pp 251ndash262 2014
[43] H Ayoglu S Gul V Hanci et al ldquoThe effects of dexmedeto-midine dosage on cerebral vasospasm in a rat subarachnoidhaemorrhage modelrdquo Journal of Clinical Neuroscience vol 17no 6 pp 770ndash773 2010
[44] T Nakano and H Okamoto ldquoDexmedetomidine-induced cere-bral hypoperfusion exacerbates ischemic brain injury in ratsrdquoJournal of Anesthesia vol 23 no 3 pp 378ndash384 2009
[45] I Tse H L Zhao and D Q Ma ldquoOrganoprotective effects ofDexmedetomidine from bench to bedsiderdquo Journal of Periop-erative Science vol 1 no 3 pp 1ndash15 2014
[46] M Tasdogan D Memis N Sut and M Yuksel ldquoResults of apilot study on the effects of propofol and dexmedetomidine oninflammatory responses and intraabdominal pressure in severesepsisrdquo Journal of Clinical Anesthesia vol 21 no 6 pp 394ndash4002009
[47] K Tanabe R Matsushima-Nishiwaki O Kozawa and HIida ldquoDexmedetomidine suppresses interleukin-1120573-inducedinterleukin-6 synthesis in rat glial cellsrdquo International Journalof Molecular Medicine vol 34 no 4 pp 1032ndash1038 2014
[48] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitstumor necrosis factor-alpha and interleukin 6 in lipopoly-saccharide-stimulated astrocytes by suppression of c-Jun N-terminal kinasesrdquo Inflammation vol 37 no 3 pp 942ndash9492014
[49] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitsinducible nitric oxide synthase in lipopolysaccharide-stimulat-ed microglia by suppression of extracellular signal-regulatedkinaserdquo Neurological Research vol 37 no 3 pp 238ndash245 2015
[50] C Chrysostomou S R Schulman M H Castellanos et al ldquoAphase IIIII multicenter safety efficacy and pharmacokineticstudy of dexmedetomidine in preterm and term neonatesrdquoTheJournal of Pediatrics vol 164 no 2 pp 276e3ndash282e3 2014
[51] K Konig and K J Guy ldquoBronchopulmonary dysplasia inpreterm infants managed with non-invasive ventilation orsurfactant and a brief period of mechanical ventilation a 6-year cohort studyrdquo The Journal of Maternal-Fetal amp NeonatalMedicine vol 27 no 6 pp 608ndash611 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Oxidative Medicine and Cellular Longevity 9
5 Conclusion
The essential core of this work is the finding that dexmedeto-midine prevents oxidative stress inflammation and celldeath in the neonatal brain under hyperoxic conditionsExposure to high oxygen in the developing brain of six-day-old rat pups leads to increased oxidative stress-induced andinflammatoryDNAdamage as demonstrated by an increasedapoptotic rate a reduction of the GSHGSSG ratio increasedlipid peroxidation and increased IL-1120573 levels Remarkably asingle dose of dexmedetomidine has weakened or abolishedall these detrimental effects under hyperoxic conditionsHowever in this study dexmedetomidine also shows negativeeffects in the control animals and these potential side effectsshould be taken into consideration in further studies on theprotection of the immature brain with dexmedetomidine
Based on these data the hyperoxic model indicatesdexmedetomidine as a sedative in pediatric anesthesia anda possible promising agent for neuroprotective strategies inpreterm infants
Conflict of Interests
The authors declare that there is no conflict of interestswith any financial organization regarding the commercialidentities mentioned in the paper
Acknowledgments
This work was supported by the Forderverein fur fruh-geborene Kinder an der Charite eV Berlin The authorsgratefully thank Evelyn Strauss and Vanessa Valdix for excel-lent technical assistance
References
[1] E Gerdin O Tyden and U J Eriksson ldquoThe developmentof antioxidant enzymatic defense in the perinatal rat lungactivities of superoxide dismutase glutathione peroxidase andcatalaserdquo Pediatric Research vol 19 no 7 pp 687ndash691 1985
[2] R E Black S Cousens H L Johnson et al ldquoGlobal regionaland national causes of child mortality in 2008 a systematicanalysisrdquoThe Lancet vol 375 no 9730 pp 1969ndash1987 2010
[3] M C Allen ldquoNeurodevelopmental outcomes of preterminfantsrdquo Current Opinion in Neurology vol 21 no 2 pp 123ndash128 2008
[4] B R Vohr ldquoNeurodevelopmental outcomes of extremelypreterm infantsrdquo Clinics in Perinatology vol 41 no 1 pp 241ndash255 2014
[5] K J S Anand and R W Hall ldquoPharmacological therapy foranalgesia and sedation in the newbornrdquo Archives of Disease inChildhood Fetal and Neonatal Edition vol 91 no 6 pp F448ndashF453 2006
[6] CMahajan andHHDash ldquoProcedural sedation and analgesiain pediatric patientsrdquo Journal of Pediatric Neurosciences vol 9no 1 pp 1ndash6 2014
[7] S G Soriano andK J S Anand ldquoAnesthetics and brain toxicityrdquoCurrent Opinion in Anaesthesiology vol 18 no 3 pp 293ndash2972005
[8] C McPherson ldquoSedation and analgesia in mechanically ven-tilated preterm neonates continue standard of care or experi-mentrdquoThe Journal of Pediatric Pharmacology andTherapeuticsvol 17 no 4 pp 351ndash364 2012
[9] R Deulofeut D Golde and S Augusto ldquoTreatment-by-gendereffect when aiming to avoid hyperoxia in preterm infants in theNICUrdquo Acta Paediatrica vol 96 no 7 pp 990ndash994 2007
[10] C J Wright and P A Dennery ldquoManipulation of gene expres-sion by oxygen a primer from bedside to benchrdquo PediatricResearch vol 66 no 1 pp 3ndash10 2009
[11] O D Saugstad M Vento S Ramji D Howard and R FSoll ldquoNeurodevelopmental outcome of infants resuscitatedwithair or 100 oxygen a systematic review and meta-analysisrdquoNeonatology vol 102 no 2 pp 98ndash103 2012
[12] U Felderhoff-Mueser P Bittigau M Sifringer et al ldquoOxygencauses cell death in the developing brainrdquo Neurobiology ofDisease vol 17 no 2 pp 273ndash282 2004
[13] M Sifringer K Genz D Brait et al ldquoErythropoietin attenuateshyperoxia-induced cell death by modulation of inflammatorymediators and matrix metalloproteinasesrdquoDevelopmental Neu-roscience vol 31 no 5 pp 394ndash402 2009
[14] M Sifringer D Brait U Weichelt et al ldquoErythropoietinattenuates hyperoxia-induced oxidative stress in the developingrat brainrdquoBrain Behavior and Immunity vol 24 no 5 pp 792ndash799 2010
[15] M Sifringer I Bendix C von Haefen et al ldquoOxygen toxicityis reduced by acetylcholinesterase inhibition in the developingrat brainrdquoDevelopmental Neuroscience vol 35 no 2-3 pp 255ndash264 2013
[16] U Felderhoff-Mueser M Sifringer O Polley et al ldquoCaspase-1-processed interleukins in hyperoxia-induced cell death in thedeveloping brainrdquo Annals of Neurology vol 57 no 1 pp 50ndash592005
[17] S Endesfelder I Zaak U Weichelt C Buhrer and T SchmitzldquoCaffeine protects neuronal cells against injury caused byhyperoxia in the immature brainrdquo Free Radical Biology andMedicine vol 67 pp 221ndash234 2014
[18] F Brehmer I Bendix S Prager et al ldquoInteraction of inflam-mation and hyperoxia in a rat model of neonatal white matterdamagerdquo PLoS ONE vol 7 no 11 Article ID e49023 2012
[19] J Ard W Doyle and A Bekker ldquoAwake craniotomy withdexmedetomidine in pediatric patientsrdquo Journal of Neurosurgi-cal Anesthesiology vol 15 no 3 pp 263ndash266 2003
[20] M A E Ramsay and D L Luterman ldquoDexmedetomidine as atotal intravenous anesthetic agentrdquo Anesthesiology vol 101 no3 pp 787ndash790 2004
[21] S M Walker R F Howard K A Keay and M FitzgeraldldquoDevelopmental age influences the effect of epidural dexmedet-omidine on inflammatory hyperalgesia in rat pupsrdquo Anesthesi-ology vol 102 no 6 pp 1226ndash1234 2005
[22] J Kuhmonen J Pokorny R Miettinen et al ldquoNeuroprotectiveeffects of dexmedetomidine in the gerbil hippocampus aftertransient global ischemiardquoAnesthesiology vol 87 no 2 pp 371ndash377 1997
[23] D Ma M Hossain N Rajakumaraswamy et al ldquoDexmedeto-midine produces its neuroprotective effect via the alpha 2A-adrenoceptor subtyperdquo European Journal of Pharmacology vol502 no 1-2 pp 87ndash97 2004
[24] O Eser H Fidan O Sahin et al ldquoThe influence of dexmedeto-midine on ischemic rat hippocampusrdquo Brain Research vol 1218pp 250ndash256 2008
10 Oxidative Medicine and Cellular Longevity
[25] M Cosar O Eser H Fidan et al ldquoThe neuroprotective effectof dexmedetomidine in the hippocampus of rabbits after sub-arachnoid hemorrhagerdquo Surgical Neurology vol 71 no 1 pp54ndash59 2009
[26] X Duan Y Li C Zhou L Huang and Z Dong ldquoDexmedeto-midine provides neuroprotection impact on ketamine-inducedneuroapoptosis in the developing rat brainrdquo Acta Anaesthesio-logica Scandinavica vol 58 no 9 pp 1121ndash1126 2014
[27] B Xiong Q-Q Shi and C-H Miao ldquoDexmedetomidinerenders a brain protection on hippocampal formation throughinhibition of nNOS-NO signalling in endotoxin-induced shockratsrdquo Brain Injury vol 28 no 7 pp 1003ndash1008 2014
[28] R M Venn J Hell and R M Grounds ldquoRespiratory effectsof dexmedetomidine in the surgical patient requiring intensivecarerdquo Critical Care vol 4 no 5 pp 302ndash308 2000
[29] J D Tobias and J W Berkenbosch ldquoSedation during mechani-cal ventilation in infants and children dexmedetomidine versusmidazolamrdquo Southern Medical Journal vol 97 no 5 pp 451ndash455 2004
[30] J D Tobias ldquoDexmedetomidine to treat opioid withdrawalin infants following prolonged sedation in the pediatric ICUrdquoJournal of Opioid Management vol 2 no 4 pp 201ndash205 2006
[31] R D Sanders J Xu Y Shu et al ldquoDexmedetomidine attenu-ates isoflurane-induced neurocognitive impairment in neonatalratsrdquo Anesthesiology vol 110 no 5 pp 1077ndash1085 2009
[32] R D Sanders P Sun S Patel M Li M Maze and D MaldquoDexmedetomidine provides cortical neuroprotection impacton anaesthetic-induced neuroapoptosis in the rat developingbrainrdquo Acta Anaesthesiologica Scandinavica vol 54 no 6 pp710ndash716 2010
[33] R R Riker Y Shehabi P M Bokesch et al ldquoDexmedetomidinevsmidazolam for sedation of critically Ill patients a randomizedtrialrdquoThe Journal of the American Medical Association vol 301no 5 pp 489ndash499 2009
[34] S M Jakob E Ruokonen R M Grounds et al ldquoDexmedeto-midine vsmidazolamor propofol for sedation during prolongedmechanical ventilation two randomized controlled trialsrdquo TheJournal of the American Medical Association vol 307 no 11 pp1151ndash1160 2012
[35] K OrsquoMara P Gal J L Ransom et al ldquoSuccessful use of dexme-detomidine for sedation in a 24-week gestational age neonaterdquoThe Annals of Pharmacotherapy vol 43 no 10 pp 1707ndash17132009
[36] K OrsquoMara P Gal J Wimmer et al ldquoDexmedetomidine versusstandard therapy with fentanyl for sedation in mechanicallyventilated premature neonatesrdquo The Journal of Pediatric Phar-macology andTherapeutics vol 17 no 3 pp 252ndash262 2012
[37] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[38] U Yis S H Kurul A Kumral et al ldquoHyperoxic exposure leadsto cell death in the developing brainrdquo Brain ampDevelopment vol30 no 9 pp 556ndash562 2008
[39] K Engelhard C Werner S Kaspar et al ldquoEffect of thealpha2-agonist dexmedetomidine on cerebral neurotransmitterconcentrations during cerebral ischemia in ratsrdquoAnesthesiologyvol 96 no 2 pp 450ndash457 2002
[40] Y Li M Zeng W Chen et al ldquoDexmedetomidine reducesisoflurane-induced neuroapoptosis partly by preserving PI3KAkt pathway in the hippocampus of neonatal ratsrdquo PLoS ONEvol 9 no 4 Article ID e93639 2014
[41] AM KaindlM Sifringer A Koppelstaetter et al ldquoErythropoi-etin protects the developing brain from hyperoxia-induced celldeath and proteome changesrdquo Annals of Neurology vol 64 no5 pp 523ndash534 2008
[42] E Koo T Oshodi CMeschter A Ebrahimnejad and G DongldquoNeurotoxic effects of dexmedetomidine in fetal cynomolgusmonkey brainsrdquo Journal of Toxicological Sciences vol 39 no 2pp 251ndash262 2014
[43] H Ayoglu S Gul V Hanci et al ldquoThe effects of dexmedeto-midine dosage on cerebral vasospasm in a rat subarachnoidhaemorrhage modelrdquo Journal of Clinical Neuroscience vol 17no 6 pp 770ndash773 2010
[44] T Nakano and H Okamoto ldquoDexmedetomidine-induced cere-bral hypoperfusion exacerbates ischemic brain injury in ratsrdquoJournal of Anesthesia vol 23 no 3 pp 378ndash384 2009
[45] I Tse H L Zhao and D Q Ma ldquoOrganoprotective effects ofDexmedetomidine from bench to bedsiderdquo Journal of Periop-erative Science vol 1 no 3 pp 1ndash15 2014
[46] M Tasdogan D Memis N Sut and M Yuksel ldquoResults of apilot study on the effects of propofol and dexmedetomidine oninflammatory responses and intraabdominal pressure in severesepsisrdquo Journal of Clinical Anesthesia vol 21 no 6 pp 394ndash4002009
[47] K Tanabe R Matsushima-Nishiwaki O Kozawa and HIida ldquoDexmedetomidine suppresses interleukin-1120573-inducedinterleukin-6 synthesis in rat glial cellsrdquo International Journalof Molecular Medicine vol 34 no 4 pp 1032ndash1038 2014
[48] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitstumor necrosis factor-alpha and interleukin 6 in lipopoly-saccharide-stimulated astrocytes by suppression of c-Jun N-terminal kinasesrdquo Inflammation vol 37 no 3 pp 942ndash9492014
[49] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitsinducible nitric oxide synthase in lipopolysaccharide-stimulat-ed microglia by suppression of extracellular signal-regulatedkinaserdquo Neurological Research vol 37 no 3 pp 238ndash245 2015
[50] C Chrysostomou S R Schulman M H Castellanos et al ldquoAphase IIIII multicenter safety efficacy and pharmacokineticstudy of dexmedetomidine in preterm and term neonatesrdquoTheJournal of Pediatrics vol 164 no 2 pp 276e3ndash282e3 2014
[51] K Konig and K J Guy ldquoBronchopulmonary dysplasia inpreterm infants managed with non-invasive ventilation orsurfactant and a brief period of mechanical ventilation a 6-year cohort studyrdquo The Journal of Maternal-Fetal amp NeonatalMedicine vol 27 no 6 pp 608ndash611 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
10 Oxidative Medicine and Cellular Longevity
[25] M Cosar O Eser H Fidan et al ldquoThe neuroprotective effectof dexmedetomidine in the hippocampus of rabbits after sub-arachnoid hemorrhagerdquo Surgical Neurology vol 71 no 1 pp54ndash59 2009
[26] X Duan Y Li C Zhou L Huang and Z Dong ldquoDexmedeto-midine provides neuroprotection impact on ketamine-inducedneuroapoptosis in the developing rat brainrdquo Acta Anaesthesio-logica Scandinavica vol 58 no 9 pp 1121ndash1126 2014
[27] B Xiong Q-Q Shi and C-H Miao ldquoDexmedetomidinerenders a brain protection on hippocampal formation throughinhibition of nNOS-NO signalling in endotoxin-induced shockratsrdquo Brain Injury vol 28 no 7 pp 1003ndash1008 2014
[28] R M Venn J Hell and R M Grounds ldquoRespiratory effectsof dexmedetomidine in the surgical patient requiring intensivecarerdquo Critical Care vol 4 no 5 pp 302ndash308 2000
[29] J D Tobias and J W Berkenbosch ldquoSedation during mechani-cal ventilation in infants and children dexmedetomidine versusmidazolamrdquo Southern Medical Journal vol 97 no 5 pp 451ndash455 2004
[30] J D Tobias ldquoDexmedetomidine to treat opioid withdrawalin infants following prolonged sedation in the pediatric ICUrdquoJournal of Opioid Management vol 2 no 4 pp 201ndash205 2006
[31] R D Sanders J Xu Y Shu et al ldquoDexmedetomidine attenu-ates isoflurane-induced neurocognitive impairment in neonatalratsrdquo Anesthesiology vol 110 no 5 pp 1077ndash1085 2009
[32] R D Sanders P Sun S Patel M Li M Maze and D MaldquoDexmedetomidine provides cortical neuroprotection impacton anaesthetic-induced neuroapoptosis in the rat developingbrainrdquo Acta Anaesthesiologica Scandinavica vol 54 no 6 pp710ndash716 2010
[33] R R Riker Y Shehabi P M Bokesch et al ldquoDexmedetomidinevsmidazolam for sedation of critically Ill patients a randomizedtrialrdquoThe Journal of the American Medical Association vol 301no 5 pp 489ndash499 2009
[34] S M Jakob E Ruokonen R M Grounds et al ldquoDexmedeto-midine vsmidazolamor propofol for sedation during prolongedmechanical ventilation two randomized controlled trialsrdquo TheJournal of the American Medical Association vol 307 no 11 pp1151ndash1160 2012
[35] K OrsquoMara P Gal J L Ransom et al ldquoSuccessful use of dexme-detomidine for sedation in a 24-week gestational age neonaterdquoThe Annals of Pharmacotherapy vol 43 no 10 pp 1707ndash17132009
[36] K OrsquoMara P Gal J Wimmer et al ldquoDexmedetomidine versusstandard therapy with fentanyl for sedation in mechanicallyventilated premature neonatesrdquo The Journal of Pediatric Phar-macology andTherapeutics vol 17 no 3 pp 252ndash262 2012
[37] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001
[38] U Yis S H Kurul A Kumral et al ldquoHyperoxic exposure leadsto cell death in the developing brainrdquo Brain ampDevelopment vol30 no 9 pp 556ndash562 2008
[39] K Engelhard C Werner S Kaspar et al ldquoEffect of thealpha2-agonist dexmedetomidine on cerebral neurotransmitterconcentrations during cerebral ischemia in ratsrdquoAnesthesiologyvol 96 no 2 pp 450ndash457 2002
[40] Y Li M Zeng W Chen et al ldquoDexmedetomidine reducesisoflurane-induced neuroapoptosis partly by preserving PI3KAkt pathway in the hippocampus of neonatal ratsrdquo PLoS ONEvol 9 no 4 Article ID e93639 2014
[41] AM KaindlM Sifringer A Koppelstaetter et al ldquoErythropoi-etin protects the developing brain from hyperoxia-induced celldeath and proteome changesrdquo Annals of Neurology vol 64 no5 pp 523ndash534 2008
[42] E Koo T Oshodi CMeschter A Ebrahimnejad and G DongldquoNeurotoxic effects of dexmedetomidine in fetal cynomolgusmonkey brainsrdquo Journal of Toxicological Sciences vol 39 no 2pp 251ndash262 2014
[43] H Ayoglu S Gul V Hanci et al ldquoThe effects of dexmedeto-midine dosage on cerebral vasospasm in a rat subarachnoidhaemorrhage modelrdquo Journal of Clinical Neuroscience vol 17no 6 pp 770ndash773 2010
[44] T Nakano and H Okamoto ldquoDexmedetomidine-induced cere-bral hypoperfusion exacerbates ischemic brain injury in ratsrdquoJournal of Anesthesia vol 23 no 3 pp 378ndash384 2009
[45] I Tse H L Zhao and D Q Ma ldquoOrganoprotective effects ofDexmedetomidine from bench to bedsiderdquo Journal of Periop-erative Science vol 1 no 3 pp 1ndash15 2014
[46] M Tasdogan D Memis N Sut and M Yuksel ldquoResults of apilot study on the effects of propofol and dexmedetomidine oninflammatory responses and intraabdominal pressure in severesepsisrdquo Journal of Clinical Anesthesia vol 21 no 6 pp 394ndash4002009
[47] K Tanabe R Matsushima-Nishiwaki O Kozawa and HIida ldquoDexmedetomidine suppresses interleukin-1120573-inducedinterleukin-6 synthesis in rat glial cellsrdquo International Journalof Molecular Medicine vol 34 no 4 pp 1032ndash1038 2014
[48] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitstumor necrosis factor-alpha and interleukin 6 in lipopoly-saccharide-stimulated astrocytes by suppression of c-Jun N-terminal kinasesrdquo Inflammation vol 37 no 3 pp 942ndash9492014
[49] X Zhang J Wang W Qian et al ldquoDexmedetomidine inhibitsinducible nitric oxide synthase in lipopolysaccharide-stimulat-ed microglia by suppression of extracellular signal-regulatedkinaserdquo Neurological Research vol 37 no 3 pp 238ndash245 2015
[50] C Chrysostomou S R Schulman M H Castellanos et al ldquoAphase IIIII multicenter safety efficacy and pharmacokineticstudy of dexmedetomidine in preterm and term neonatesrdquoTheJournal of Pediatrics vol 164 no 2 pp 276e3ndash282e3 2014
[51] K Konig and K J Guy ldquoBronchopulmonary dysplasia inpreterm infants managed with non-invasive ventilation orsurfactant and a brief period of mechanical ventilation a 6-year cohort studyrdquo The Journal of Maternal-Fetal amp NeonatalMedicine vol 27 no 6 pp 608ndash611 2014
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
Submit your manuscripts athttpwwwhindawicom
Stem CellsInternational
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
MEDIATORSINFLAMMATION
of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Behavioural Neurology
EndocrinologyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Disease Markers
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
BioMed Research International
OncologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Oxidative Medicine and Cellular Longevity
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
PPAR Research
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
ObesityJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Computational and Mathematical Methods in Medicine
OphthalmologyJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Diabetes ResearchJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Research and TreatmentAIDS
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Gastroenterology Research and Practice
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Parkinsonrsquos Disease
Evidence-Based Complementary and Alternative Medicine
Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom
