Sevoflurane Preconditioning Induces.

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 ischemia–reperfusion injury through up-regulating antioxidant enzyme activities before ischemic injury bygenerating reactive oxygen species (ROS).METHODS: In preconditioning groups, adult male Sprague–Dawley 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 dose–response 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;112:931–7)

The neuroprotective effects of anesthetic precondi-tioning have been reported in both in vitro and invivo models.1–4 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 ischemia–reperfusion 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 neuronal–glial 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 oxidant–antioxidant mecha-nisms of this neuroprotection. We hypothesized thatsevo preconditioning could dose-dependently reducecerebral ischemia–reperfusion 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, Xi’an, 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, Xi’an 710032,Shaanxi Province, China. Address e-mail [email protected].

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 (Xi’an, China). MaleSprague–Dawley 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 at21°C � 2°C, 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 ischemia–reperfusion 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 sevo � NAC 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. DMTU was 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, rats’neurologic 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 has beendescribed previously.12,14,15 The infarct volume was calcu-lated by Swanson and Sharp’s method to correct for edema:100 � (contralateral hemisphere volume – nonlesionedipsilateral hemisphere volume)/contralateral hemispherevolume.17

Antioxidant Enzyme Activity MeasurementSamples of the ischemic right hemisphere were homoge-nized in cold saline with a weight-to-volume ratio of 1:10.The homogenate was centrifuged at 3000 revolutions perminute (rpm) for 15 minutes at 4°C, and the supernatantwas removed to a cuvette stored at �80°C. Serum wasisolated after blood samples (1 mL) had been centrifuged,and frozen at �80°C until analysis was performed. Themeasurements of antioxidant enzyme activity were per-formed according to the technical manuals of the detectionkits.11 The activity of superoxide dismutase (SOD), catalase(CAT), and glutathione peroxidase (GSH-px) were respec-tively surveyed by measuring the absorbance at 550, 240,and 412 nm by using an ultraviolet light spectrophotometer(DU800, Beckman Coulter, Brea, CA).

Statistical AnalysisThe software SPSS 11.0 for Windows was used to conductstatistical analyses. All values, except for neurologic scores,were presented as mean � sd (SD), and analyzed by 1-wayanalysis of variance (ANOVA). Between-groups differenceswere detected with a Tukey post hoc test. The neurologicalscores, presented as median (interquartile range), were

Sevoflurane Induces Neuroprotection via Reactive Oxygen Species

932 www.anesthesia-analgesia.org ANESTHESIA & ANALGESIA

analyzed by a nonparametric method (Kruskal–Wallis test)followed by the Mann–Whitney U test with Bonferronicorrection. Correlations of the serum antioxidant levels andthe percentages of brain infarct volumes were analyzed byusing Spearman’s rank correlation test. A P value of �0.05was considered to be statistically significant.

RESULTSPhysiologic ParametersPhysiologic variables at the end of the last exposure andduring surgery are summarized in Table 1. At the end ofthe last preconditioning, animals treated with 4% sevo hadslight respiratory depression (pH 7.3 � 0.1; Po2 201.4 � 6.5mm Hg; Pco2 56.0 � 1.5 mm Hg), without hypotension orhypothermia.

The initial CBF before occlusion was recorded as 100%,and subsequent flow changes were expressed in relation tothis value. During occlusion, the CBF values remained at

�30% of baseline for all rats. At the onset of reperfusion,CBF recovered up to �70% of baseline, and then returnedto baseline within 30 minutes (Fig. 1).

Sevoflurane Preconditioning Dose-DependentlyInduces NeuroprotectionThe neurobehavioral outcomes of groups 4% sevo and 2%sevo were better than those of the control, vehicle, and 1%sevo groups (P � 0.001, for both 4% sevo 14.5 [4.25] and2% sevo 12.5 [4.5] vs. control 9 [2.5]; P � 0.005, 4% sevovs. 1% sevo 10 [3.25]; P � 0.029, 2% sevo vs. 1% sevo) at72 hours after reperfusion. Four percent sevo also in-duced better neurologic behavior than did 2% sevo (P �0.044, Fig. 2A).

Two percent sevo and 4% sevo reduced the brain infarctsize at 72 hours after stroke (P � 0.035, 2% sevo 37.2 � 5.9%vs. vehicle 45.8 � 6.3%; P � 0.001, 4% sevo 29.2 � 6.7% vs.vehicle). Brain infarct volume percentages in the 4% sevogroup were even lower than that in the 1% sevo group(44.4 � 6.0%, P � 0.003, Fig. 2B). With respect to respiratorydepression induced by 4% sevo, we chose 2% as thepreconditioning concentration in subsequent experiments.

Neuroprotective Effect of SevofluranePreconditioning Is Reversed by DMTU and NACAs is shown in Figure 3A, sevo preconditioning improvedneurologic behavior scores. This beneficial outcome wasreversed by preconditioning with DMTU and NAC (P �0.036 and 0.024, for sevo � DMTU 9 [4.25] and sevo � NAC9.5 [3.5] vs. sevo 12 [3.5], respectively). Infarct sizes ofsevo � DMTU and sevo � NAC groups were larger thanthose of sevo groups (P � 0.035 and 0.036, for sevo �DMTU 47.6 � 5.3% and sevo � NAC 48.6 � 5.0% vs. sevo38.5 � 4.9%, respectively, Fig. 3B), but DMTU and NAChad no such effects on brain ischemic outcomes whenadministered alone.

Figure 1. Regional cerebral bloodflow of ischemic hemisphere dur-ing surgery. Control � animals suffered from middle cerebral arteryocclusion (MCAO) without any preconditioning; preconditioning �animals received 100% oxygen, 1%, 2%, or 4% sevoflurane precon-ditioning. Data are expressed as mean � SD.

Table 1. Physiologic Variables

MAP Temperature (°C) pHPaO2

(mm Hg)PaCO2

(mm Hg)End of anesthetic preconditioning

Control 100.2 � 1.1 37.5 � 0.1 7.40 � 0.02 103.0 � 0.6 37.1 � 0.2Vehicle 102.0 � 1.0 37.2 � 0.1 7.40 � 0.05 230.0 � 1.3* 35.0 � 0.31% sevo 103.4 � 1.5 37.6 � 0.1 7.39 � 0.03 229.7 � 2.7* 35.2 � 0.62% sevo 102.8 � 1.8 37.6 � 0.1 7.37 � 0.06 227.5 � 2.2* 42.5 � 0.44% sevo 100.2 � 2.1 36.7 � 0.2 7.30 � 0.07 201.4 � 6.5* 56.0 � 1.5*†

Onset of MCAOControl 100.2 � 1.0 37.0 � 0.1 7.40 � 0.03 100.0 � 0.9 39.4 � 1.6Vehicle 102.0 � 0.9 37.1 � 0.2 7.40 � 0.05 104.0 � 0.8 38.1 � 1.91% sevo 103.3 � 1.5 37.3 � 0.2 7.39 � 0.04 104.7 � 1.2 38.3 � 2.22% sevo 101.5 � 1.9. 37.2 � 0.1 7.40 � 0.06 102.5 � 1.3 39.7 � 2.34% sevo 103.0 � 1.6 37.0 � 0.2 7.39 � 0.05 106.1 � 1.5 38.2 � 2.6

Onset of reperfusionControl 99.0 � 0.9 36.9 � 0.1 7.38 � 0.07 103.0 � 0.8 39.5 � 1.9Vehicle 101.0 � 1.5 37.1 � 0.2 7.39 � 0.04 110.1 � 1.2 39.6 � 2.31% sevo 101.1 � 1.7 37.3 � 0.2 7.39 � 0.03 103.4 � 1.3 38.1 � 2.52% sevo 102.0 � 1.8 37.2 � 0.2 7.40 � 0.05 109.8 � 1.4 38.2 � 2.44% sevo 102.3 � 2.3 36.9 � 0.2 7.39 � 0.06 106.4 � 1.5 39.8 � 2.8

Data represent mean � SD (n � 5).Vehicle group, animals received 100% oxygen vehicle, 1 hour/day for 5 days; 1% sevo, 2% sevo, and 4% sevo groups received 1%, 2%, and 4% sevoflurane in100% oxygen 1 hour/day for 5 days, respectively.MAP � mean arterial blood pressure; MCAO � middle cerebral artery occlusion.* P � 0.05 vs. control. † P � 0.05 vs. 2% sevo.


Antioxidant Enzyme Activities in Brain TissueBefore IschemiaAt 24 hours after the last pretreatment, activities of totalSOD (T-SOD) in the right hemispheres in the sevo and O2

vehicle groups were higher than that of the control group(P � 0.001 and P � 0.011 for sevo (4571.9 � 502.6) and O2

vehicle (4363.0 � 427.1) vs. control (3654.8 � 318.6), respec-tively), and were decreased by NAC (P � 0.001, for bothvehicle vs. vehicle � NAC (3333.0 � 398.2) and sevo vs.sevo � NAC (3471.0 � 418.2) (Fig. 4A)).

Higher activities of copper (Cu), zinc (Zn)-SOD werefound in sevo and vehicle groups (P � 0.001 and 0.001, forsevo (3075.6 � 466.3) and vehicle (2885.4 � 251.1) vs.control (2258 � 208.7), respectively), and were reduced byNAC (P � 0.001, for vehicle vs. vehicle � NAC 2096 � 98.5and sevo vs. sevo � NAC 2159.3 � 185.8, Fig. 4B). How-ever, the activity of manganese (Mn)-SOD was not statisti-cally different among the 5 groups (Fig. 4C).

CAT activity in the sevo group (1.1 � 0.1) was higherthan that in the control (0.7 � 0.1, P � 0.001), which wasdecreased by NAC (0.6 � 0.1, P � 0.001, Fig. 4D).

GSH-px activity in the sevo groups (951.1 � 96.8) washigher than that in the control (704.9 � 100.4, P � 0.001)and vehicle (855.8 � 75.6, P � 0.001) groups, and was

significantly decreased in the sevo � NAC group (540.1 �77.5, P � 0.001, Fig. 4E).

On the basis of the above results, CAT activity and GSH-px activity, which was increased in the sevo precondi-tioning groups, were chosen to be further tested in serum.

Serum Antioxidant ActivitiesAs is shown in Figure 5A, no significant CAT activity wasobserved in serum among the control, vehicle, and sevogroups (Fig. 5A). However, serum GSH-px activity in-creased significantly in sevo groups (sevo 211.1 � 29.3 vs.control 172.6 � 30.2, P � 0.039; Fig. 5B).

Additionally, serum GSH-pX activity negatively corre-lated with the infarct volume of ischemic brain (correlationcoefficient � �0.73, 95% confidence interval [CI](�1.36–�0.51), Fig. 5C).

DISCUSSIONAnesthetic preconditioning may prevent or delay neuro-logical complications such as perioperative brain ischemicinjury, which can occur during or after surgical procedures,such as carotid endarterectomy and aortic repair.18 The opti-mal dose to maximize neuroprotection is not clearly estab-lished for sevo, a potent neuroprotective drug, and varies

Figure 2. Neurologic behavior scores (A)and infarct volume percentages (B) ofrats preconditioned with different dosesof sevoflurane. Sevoflurane (sevo) pre-conditioning improved neurologic scoresand reduced brain infarct volumes in adose-dependent manner. Neurologic be-havior scores represent median (inter-quartile range); the infarct volumepercentages are expressed as median(horizontal bars) with the individual val-ues. *P � 0.01 in comparison with thecontrol group and 1% sevo group; #P �0.05 in comparison with 2% sevo group.

Figure 3. Neurologic behavior scores (A) and infarct volume percentages (B) of rats after reperfusion. Dimethylthiourea (DMTU) andN-acetylcysteine (NAC) reversed neurobehavioral recovery and the reduction of brain infarct volumes induced by sevoflurane preconditioning.Vehicle � 100% oxygen vehicle preconditioning; DMTU � vehicle preconditioning with dimethylthiourea administration; NAC � vehiclepreconditioning with N-acetylcysteine administration; sevo � sevoflurane preconditioning; sevo � DMTU � sevoflurane preconditioning withdimethylthiourea administration; sevo � NAC � sevoflurane preconditioning with N-acetylcysteine administration. Neurologic behavior scoresrepresent median (interquartile range); the infarct volume percentages are expressed as median (horizontal bars) with the individual values.*P � 0.05 in comparison with control and vehicle; #P � 0.05 in comparison with sevo group.



widely in the literatures. The sevo preconditioning protocoland dose gradient used in this study were chosen on the basisof previous reports.14,19 In the present study, we verified that2% sevo pretreatment was sufficient to reduce infarct volumeand neurologic deficits, whereas 1% was not, which suggeststhat a threshold concentration of sevo is needed to induceischemic tolerance. Although 4% sevo preconditioning in-duced a better neuroprotective outcome than did 2% sevo, italso contributed to hypercapnia at the end of preconditioning.Hence, it is more reasonable to use 2% to study the mecha-nisms of sevo preconditioning-induced neuroprotection.

As has been shown, sevo preconditioning-induced tol-erance to focal cerebral ischemia–reperfusion injury wasreversed by DMTU and NAC. DMTU, a potent free-radicalscavenger, mainly scavenges hydroxyl radicals20,21; NAC, aGSH-reducing substrate, is widely used as an antioxidantto eliminate lipid peroxidation and H2O2.22 This paradigmwith low dosages was supposed to reduce the ROS inducedby preconditioning while not affecting the ROS producedby subsequent ischemia–reperfusion injury.12,13 Our resultsstrongly suggest that an initial oxidative stress generatedby sevo preconditioning may trigger cascades that finallylead to ischemic tolerance. This is also consistent withprevious publications that the ROS level was elevatedimmediately after isoflurane preconditioning.23,24

Thereafter, our results showed that sevoflurane precon-ditioning induced an increase in activities of T-SOD, Cu,Zn-SOD, CAT, and GSH-px in the brain tissue. NACreduced the elevation of antioxidant enzyme activity,which illustrates a direct relationship between ROS genera-tion and the increase of antioxidant enzyme activity aftersevo preconditioning.

However, in the current study, the increase of T-SODand Cu, Zn-SOD activity was reasonably attributed to theeffects of O2 vehicle, because there were no differencesbetween the sevo and control groups. Thus, CAT andGSH-px may play much more important roles than doother antioxidant enzymes in brain ischemic toleranceinduced by sevo preconditioning. In previous studies, therewas no consistency in the types of increased antioxidantenzymes after different preconditioning. The increase ofCu, Zn-SOD and GSH-px activity was identified afterhypoxic preconditioning by Arthur et al.,25 but they failedto identify the increase in CAT activity after hypoxicpreconditioning. The up-regulation of CAT and SOD, butnot GSH-px, was found in our previous study after hyper-baric oxygen preconditioning. It has also been shown thatMn-SOD activity was increased by some precondition-ing.26,27 The discrepancies between our current results andthe previous findings could be due to the use of differenttypes of animal models or preconditioning stimuli.

In the serum study, GSH-px activity was significantlyincreased by sevo rather than CAT, which could be ex-plained in 2 ways: first, the oxidation–antioxidation systemin the periphery and central nervous system may havedifferent sensitivities to anesthetic preconditioning. Sec-ond, the normal concentration of GSH-px in blood is muchhigher than that of CAT and may be much easier to detectwhen it changes. However, these conjectures should beverified by further experiments.

Figure 4. The activity of antioxidant enzymes in brain tissue at 24hours after preconditioning. The activity of total superoxidedismutase (T-SOD) (A), Cu, Zn-SOD (B), catalase (CAT) (D), andglutathione peroxidase (GSH-px) (E), except for Mn-SOD (C), wereup-regulated after oxygen and sevoflurane preconditioning. Only CATand GSH-px were exclusively increased by sevoflurane. *P � 0.05 incomparison with control group. #P � 0.05 in comparison withcorresponding preconditioning group. †P � 0.05 in comparison withvehicle group. Data are expressed as mean � SD (n � 15).

Figure 5. Serum antioxidant enzyme activities and correlation be-tween GSH-px activity with infarct volume. Serum glutathione peroxi-dase (GSH-px) activity (B), but not catalase (CAT) activity (A) wasexclusively increased by sevoflurane preconditioning before isch-emia. Serum GSH-px level also correlated well with infarct volumepercentage after stroke (C). *P � 0.05 vs. control. Correlationcoefficient � �0.73, 95% confidence interval (�1.36–�0.51), P �0.001. Data are expressed as mean � SD (n � 12).


Serum GSH-px activity conferred by sevo precondition-ing before brain ischemia had a close negative relationshipwith infarct volume percentage after reperfusion, which pro-vides a potential method to predict the efficiency of sevopreconditioning before the advent of stroke lesion. This resultwas in line with the previous finding that GSH-px activity inerythrocytes of stroke-prone spontaneously hypertensive ratsdecreased significantly at the onset of stroke, and was shownto be useful as an index for judging the progress of strokebiochemically.28 Even so, future studies should be performedto develop GSH-px to be a candidate indicator for predictingthe effectiveness of anesthetic preconditioning and the prog-nosis of stroke lesions.

Several limitations of this study deserve comment. First,among 4 antioxidant enzymes in brain tissue, which we testedbefore ischemia, there was no difference in T-SOD and its 2subtypes between the sevo and vehicle preconditioninggroups. Because only 1 time point (24 hours after last precon-ditioning) was tested in our study, we cannot exclude thepossibility that SOD participates in the preconditioning effectof sevo. Therefore, the time course of antioxidant enzymechanges after sevo preconditioning during normoxia deservesfurther study. Second, the ROS-mediated signaling cascadesin sevo preconditoning are quite complicated. One mecha-nism proposed in Riess et al.’s study of sevo preconditioningon guinea pig isolated hearts was that ROS was related to thechanges in the KATP channel, an important agent involved inanesthetic preconditioning neuroprotection.29 We have alsoevaluated previously the results of the role of KATP in isoflu-rane preconditioning.14 The relationship between ROS andKATP in the mechanisms of sevo preconditioning in the ratbrain needs further elucidation.

In conclusion, this study demonstrates for the first timethat sevo preconditioning induced cerebral ischemic toler-ance in a dose-dependent manner. DMTU and NAC, 2potent free-radical scavengers, reversed the neuroprotec-tion of sevo preconditioning. NAC also abolished theincrease of antioxidant enzyme activity induced by sevoexposure in brain tissue before ischemia. The increasedactivity of GSH-px in serum conferred by sevo was nega-tively correlated to the degree of ischemic injury in terms ofinfarct volumes. These data indicate that sevo precondi-tioning induces cerebral ischemic tolerance in a dose–response manner through ROS-mediated up-regulation ofantioxidant enzyme activities in rats. Our data support theidea that in the future, it might be possible to use serumGSH-px activity as a marker to assess the effectiveness ofsevo preconditioning before high-risk surgeries, such ascarotid endarterectomy or repair of the ascending aorta, forthe alleviation of perioperative cerebral ischemic injury.18

ACKNOWLEDGMENTSWe thank Dr. Yan Lu (Department of Anesthesiology, XijingHospital, Fourth Military Medical University) for his criticalreading of the current version of the manuscript.

DISCLOSURESName: Qianzi Yang, MD.Contribution: Conduct of study, data interpretation, andmanuscript preparation.Name: Hui Dong, MD, PhD.

Contribution: Study design and manuscript preparation.Name: Jiao Deng, MD.Contribution: Conduct of study.Name: Qiang Wang, MD, PhD.Contribution: Manuscript preparation.Name: Ruidong Ye, MD.Contribution: Conduct of study.Name: Xuying Li, MD.Contribution: Data analysis.Name: Sheng Hu, MD, PhD.Contribution: Data analysis.Name: Hailong Dong, MD, PhD.Contribution: Manuscript review.Name: Lize Xiong, MD, PhD.Contribution: Study design, data interpretation, and manu-script review.

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Sevoflurane Preconditioning Induces.

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