Sevoflurane Preconditioning Induces NeuroprotectionThrough Reactive Oxygen Species-MediatedUp-Regulation of Antioxidant Enzymes in RatsQianzi Yang, MD, Hui Dong, MD, PhD, Jiao Deng, MD, Qiang Wang, MD, PhD, Ruidong Ye, MD,Xuying Li, MD, Sheng Hu, MD, PhD, Hailong Dong, MD, PhD, and Lize Xiong, MD, PhD
BACKGROUND: It has been reported that sevoflurane preconditioning can induce neuroprotec-tion, the mechanisms of which, however, are poorly elucidated. We designed the present studyto examine the hypothesis that sevoflurane preconditioning could reduce cerebral ischemiareperfusion injury through up-regulating antioxidant enzyme activities before ischemic injury bygenerating reactive oxygen species (ROS).METHODS: In preconditioning groups, adult male SpragueDawley rats were pretreated with 1hour sevoflurane exposure at a dose of 1%, 2%, or 4% for 5 consecutive days. At 24 hours afterthe last exposure, all rats were subjected to focal brain ischemia induced by middle cerebralartery occlusion for 120 minutes followed by 72-hour reperfusion. The role of ROS in ischemictolerance was assessed by administration of the free radical scavenger dimethylthiourea andantioxidant N-acetylcysteine before each preconditioning. Brain ischemic injury was evaluated byneurologic behavior scores and brain infarct volume calculation. Antioxidant enzyme activities(superoxide dismutase, catalase, and glutathione peroxidase [GSH-px]) of brain tissue and bloodserum were tested at 24 hours after the last sevoflurane preconditioning.RESULTS: Sevoflurane preconditioning reduced infarct size and improved neurobehavioraloutcome in a dose-dependent manner. The neuroprotective effects of sevoflurane precondition-ing were abolished by dimethylthiourea and N-acetylcysteine. The activities of catalase andglutathione peroxidase (GSH-px) in the brain tissue were elevated by sevoflurane preconditioningbefore ischemic injury. The up-regulated activity of GSH-px in serum negatively correlated withbrain infarct volume percentage.CONCLUSION: Sevoflurane preconditioning induces cerebral ischemic tolerance in a doseresponse manner through ROS release and consequent up-regulation of antioxidant enzymeactivity before ischemic injury in rats. Serum GSH-px activity could be developed as a marker toassess the effectiveness of sevoflurane preconditioning before ischemia. (Anesth Analg 2011;112:9317)
The neuroprotective effects of anesthetic precondi-tioning have been reported in both in vitro and invivo models.14 Sevoflurane (sevo), a popular anes-thetic with few clinical side effects, has been shown toreduce cerebral ischemia damage in various experimentalmodels. However, the dose-dependent effects of sevo pre-conditioning have not been evaluated, and its mechanismsare poorly elucidated.
Nonlethal levels of reactive oxygen species (ROS) gen-eration have been proposed to be initiators or mediators forischemic tolerance induction.5 It has been demonstratedthat the cardioprotective effects of sevo against ischemiareperfusion injury required ROS generation. ScavengingROS abolished the preconditioning effects of anesthetics by
attenuating mitochondrial uncoupling6 or protein kinases.7
In the central nervous system, ROS was also involved inneuroprotection from sevo preconditioning in mixed corti-cal neuronalglial cell cultures against oxygen and glucosedeprivation.8 In addition, several studies have shown thatendogenous antioxidant enzymes could be induced simul-taneously with the development of neuroprotection bydifferent preconditioning methods such as lipopolysaccha-ride9 and brief ischemia.10 Our previous study on spinalcord ischemia in a rabbit model has demonstrated thathyperbaric oxygen preconditioning activates endogenousantioxidant enzymes through generation of a small amountof ROS.11
Therefore, the current study was designed to test thedose-dependent neuroprotective effects of sevo precon-ditioning in a focal cerebral ischemia model in rats, andfurthermore to verify the oxidantantioxidant mecha-nisms of this neuroprotection. We hypothesized thatsevo preconditioning could dose-dependently reducecerebral ischemiareperfusion injury through releasingROS, which up-regulates protective antioxidant enzymesbefore ischemic injury.
METHODSThe experimental protocol used in this study was approvedby the Ethics Committee for Animal Experimentation of theFourth Military Medical University and was conducted
From the Department of Anesthesiology, Xijing Hospital, Fourth MilitaryMedical University, Xian, Shaanxi, China.
Accepted for publication November 16, 2010.
Funding: This work was supported by the National Science Fund forDistinguished Young Scholars (grant 30725039) and the Major Program ofNational Natural Science Foundation of China (grant 30930091).
Drs. Qianzi Yang and Hui Dong contributed equally to this work.
The authors declare no conflict of interest.
Reprints will not be available from the authors.
Address correspondence to Lize Xiong, MD, PhD, Department of Anesthe-siology, Xijing Hospital, Fourth Military Medical University, Xian 710032,Shaanxi Province, China. Address e-mail firstname.lastname@example.org.
Copyright 2011 International Anesthesia Research SocietyDOI: 10.1213/ANE.0b013e31820bcfa4
April 2011 Volume 112 Number 4 www.anesthesia-analgesia.org 931
according to the Guidelines for Animal Experimentation ofthe Fourth Military Medical University (Xian, China). MaleSpragueDawley rats (280 to 300 g, 10-weeks-old) wereprovided by the Experimental Animal Center of the FourthMilitary Medical University and housed under controlledcondition with a 12-hour light/dark cycle, temperature at21C 2C, and humidity in 60%70% for at least 1 weekbefore drug treatment or surgery.
Experimental ProtocolExperiment 1. To assess the dose-dependent neuroprotec-tive effects of sevo preconditioning on cerebral ischemiareperfusion injury, we randomly assigned 50 rats to 5groups: control group; vehicle group, which received 100%oxygen for 1 hour per day; and sevo groups, whichreceived 1%, 2%, or 4% sevo in 100% oxygen for 1 hour perday (Baxter, Guayama, Puerto Rico). Both vehicle and sevowere given for 5 consecutive days. At 24 hours after the lastpreconditioning, all rats were subjected to middle cerebralartery occlusion (MCAO).Experiment 2. To evaluate the involvement of the initialoxidative stress in the development of ischemic toleranceinduced by sevo preconditioning, 70 rats were randomlyassigned to 7 groups: control, vehicle, dimethylthiourea(DMTU), N-acetylcysteine (NAC), sevo, sevo DMTU,and sevo NAC. Animals in groups sevo, sevo DMTU,and sevoNAC were exposed to sevo. The dosage of sevoexposure was screened from experiment 1; vehicle, DMTU,and NAC groups received oxygen preconditioning. Boththe free-radical scavenger DMTU and antioxidant NACwere dissolved in saline. DMTUwas administered at a doseof 500 mg/kg (i.p.), 1 hour before preconditioning, whereasNAC was supplied at 150 mg/kg (i.p.), 30 minutes inadvance (Sigma-Aldrich, St. Louis, MO).12,13 Vehicle andsevo groups received the same volume of saline vehicle.Experiment 3. To study the mechanisms of changes ofantioxidant enzyme activity after sevo preconditioning, thisexperiment consisted of 2 parts. In part 1, 75 rats weregrouped into control, vehicle, vehicle NAC, sevo, andsevo NAC. At 24 hours after the last preconditioning, allrats were decapitated to evaluate enzyme activity of theright hemisphere (detection kits, Jianchen Biological Insti-tute, Nanjing, China). In part 2, 36 rats were grouped intocontrol, vehicle, and sevo. Blood samples of animals wereobtained from femoral arteries to evaluate serum enzymeactivity at 24 hours after the last preconditioning. The samevolume of warm normal saline was infused through thevena caudalis. Animals in part 2 were then subjected toMCAO.
Sevoflurane PreconditioningAll rats were acclimated to the animal room for 1 week.Rats in the preconditioning groups were put in a transpar-ent chamber comprising an airtight box (50 40 30 cm3)with a gas inlet port and an outlet port. During precondi-tioning, inspired and expired fractions of sevo, oxygen, andcarbon dioxide were continuously monitored (MP-60, Phil-lips Medical Systems, Best, The Netherlands). Carbon di-oxide was cleared by using soda lime (Molecular ProductsLimited, Essex, UK) at the bottom of the container.
Transient Focal Cerebral IschemiaFocal cerebral ischemia was induced by MCAO in ratsusing an intraluminal filament technique, as was describedpreviously.14,15 Regional cerebral bloodflow (rCBF) wasmonitored through a disposable microtip fiberoptic probe(diameter 0.5 mm) connected through a Master Probe to alaser Doppler computerized main unit (PeriFlux 5000,Perimed AB, Stockholm, Sweden). MCAO was consideredadequate if rCBF sharply decreased to 30% of the baseline(preischemia) level; otherwise, animals were excluded fromanalysis. Reperfusion was accomplished by removing thesuture after 120 minutes of ischemia, and wounds weresutured.
Arterial Blood Gas DeterminationArterial blood was taken from 20 rats, which were groupedtogether in experiment 1 (n 5 for each group). A catheterwas inserted to the left femoral artery to draw the bloodsample. About 0.3 mL of blood per rat was taken via thefemoral artery at the end of the last exposure, onset ofMCAO, and reperfusion. Blood gas was immediately ana-lyzed (Rapidlab 1260, Bayer HealthCare, Uxbridge, UK).
Neurobehavioral Evaluation andInfarct AssessmentAt 24 hours, 48 hours, and 72 hours after reperfusion, ratsneurologic behaviors were assessed by an observer whowas blind to animal groups, according to the method ofGarcia et al.16 Animals were then decapitated, and 2-mm-thick coronal sections throughout the brain were stainedwith 2% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma,St. Louis, MO) to evaluate the infarct volume, as