Journal of Surgical Research 160, 53–59 (2010)doi:10.1016/j.jss.2008.12.034

Delayed Cardioprotective Effects of Hyperoxia Preconditioning Prolonged

by Intermittent Exposure

Babak Baharvand, M.D.,jj Mansour Esmaili Dehaj, Ph.D.,*,§,1 Mohsen Foadaddini, M.Sc.,‡Bahram Rasoulian, M.D., Ph.D.,#,** Khalil Poorkhalili, Ph.D.,{ Hannaneh Wahhab Aghai, Ph.D.,‡

and Ali Khoshbaten, Ph.D.†,‡

*Research Center for Trauma, †Research Center for Chemical Injuries, ‡Department of Physiology and Biophysics, Baqiyatallah University ofMedical Sciences, Tehran, Iran; §Department of Physiology, Yazd University of Medical Sciences, Yazd, Iran; jjDivision of heart in Shohadaye

ashayer hospital of Lorestan University of Medical Sciences, Khoramabad, Iran; {Department of Physiology, School of Medical Sciences,Boohsher University of Medical Sciences, Tehran, Iran; #Department of Physiology, Faculty of Medicine, Lorestan University

(Medical Sciences), Khoramabad, Iran; and **Razi Herbal Medicines Research Center and Department of Physiology, Lorestan University(Medical Sciences), Khoramabad, Iran

Submitted for publication July 21, 2008

Background. In our previous study, it was indicatedthat pre-exposing rats to normobaric hyperoxia couldinduce a late preconditioning against infarction andarrhythmia. In this study, attempts were made toknow whether the intermittent pre-exposure to thesame environment could prolong the late phase of hy-peroxia preconditioning.

Methods. In the first series of experiments, rats weredivided into five groups; group 1 was pre-exposed tonormal air (NOR) and the other groups to hyperoxicair (O2 > 95%, 120 min once a d) 12, 24, 48, and 72 h(H12, H24, H48, and H72 groups) before 30 min ische-mia. In the second series of experiments, rats werepre-exposed to intermittent hyperoxic air (1, 2, or 3 con-secutive d) at different times before being subjected toischemia (H48, H2-48, H2-72, H3-72, and H3-96 groups).The infarct size was measured by triphenyltetrazoliumchloride staining, and lead II of electrocardiogram re-corded to monitor ischemic-induced arrhythmia.

Results. Compared with NOR group, the infarct sizeand incidence of arrhythmia were reduced signifi-cantly in H24 and H48 groups. When the exposure pe-riods were enhanced to 2 d, the infarct size did notdecrease significantly, but the incidence of arrhythmiareduced. When the pre-exposure times were enhancedto 3 d, both the infarct size and incidence of arrhyth-mia decreased significantly in H3-72 group, but not inH3-96 group.

1 To whom correspondence and reprint requests should be ad-dressed at Department of Physiology, Faculty of Medicine, Yazd Uni-versity of Medical Sciences, P. O. Box 19395/6558. Yazd, Iran. E-mail:med1354@yahoo.com.

53

Conclusion. These results show that the late phaseof hyperoxia preconditioning may last for more than48 h and prolong by intermittent per-exposure to thesame environment. � 2010 Elsevier Inc. All rights reserved.

Key Words: hyperoxia; preconditioning; intermit-tent; infarct; arrhythmia.

INTRODUCTION

Ischemic-reperfusion (I/R) injury is a major complica-tion occurring in heart stroke, coronary artery revascu-larization, and heart transplantation [1, 2]. Reactiveoxygen species (ROS) were initially thought to playa role in the pathogenesis of I/R injury [2–7]. Recentstudies involving ischemic preconditioning (IPC) haveidentified ROS as potential mediators for the cardiopro-tective effects observed following this phenomenon, aswell as other mediators, like adenosine, nitric oxide,etc. [3–6].

Likewise, under normal physiological conditions, 1%to 4% of available oxygen is converted to ROS. ROS levelis increased to higher levels in proportion to dose-depen-dent of oxygen concentration [8]. Several studies havereported that transient pre-exposure to high levels of ox-ygen concentrations (O2> 90 %), as a low-graded sys-temic oxidative stress stimulus, could inducepreconditioning-like effects in heart [9–11], brain [12,13], spinal cord [14, 15], and liver [16], which are compa-rable to the effects of IPC. Our colleagues have also

0022-4804/08 $36.00� 2010 Elsevier Inc. All rights reserved.

JOURNAL OF SURGICAL RESEARCH: VOL. 160, NO. 1, MAY 1, 201054

shown that pre-exposure to normobaric hyperoxia couldinduce protective effects on rat kidney and brain [17,18]. Previously, we have also shown that pre-exposingrats to normobaric hyperoxia (O2> 95%) for at least120 min protects the anesthetized rat hearts against in-farction and ischemic arrhythmia in a time- and concen-tration-dependent manner 24 h later [19]. This isconsistent with the works of Tahepold’s group using iso-lated rat and mouse hearts [20, 21].

The purpose of this study was to investigate (1) howlong does the late phase of hyperoxic preconditioninglast in the heart of anesthetized rats and to determine(2) whether the intermittent normobaric hyperoxia(pre-exposure to the same hyperoxic environment 24 hprior to the termination of the previous late phase of hy-peroxic preconditioning) can potentiate and prolongthis phase.

MATERIAL AND METHODS

Animals

Male Wistar rats (250–300 g) were housed at 12:12-h light-dark cy-cles inside Baqiyatallah University animal house. They were fed adlibitum and conditioned in this nonstressful environment for at least1 wk. The study was performed in accordance with the Guide for theCare and Use of Laboratory Animals published by the National Insti-tutes of Health, and was approved by the ethics committee for animalresearch at Baqiyatallah University.

Surgical Preparation

Rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.).Rectal temperature was monitored and maintained at 37.5 �C. Theneck was opened with a ventral midline incision and tracheotomywas performed, followed by intubation. The animals were artificiallyventilated at 80 strokes/min with a tidal volume of 1 mL/100 g tomaintain PO2, PCO2, and pH in the normal physiological range.The tail vein was cannulated with an angiocat (yellow color) for theinfusion of Evans blue solution. Left carotid artery, likewise, wasdissected and cannulated for continuous arterial pressure monitor-ing. Electrocardiographic leads were attached to subcutaneouselectrodes to monitor limb lead II continuously. A left thoracotomywas performed in the fourth intercostal space and the pericardiumwas opened to expose the heart. A 5-0 silk suture with an atraumaticneedle was then passed around the left anterior descending coronaryartery (LAD), midway between the atrioventricular groove andthe apex, and a snare formed by passing both ends of the suturethrough a piece of polyethylene tube. After 15 to 20 min stabilization,LAD was occluded by clamping the snare against the surface of theheart (30 min). Ischemia was confirmed by the regional cyanosisdownstream of the occlusion, by the ST elevation, and by the reducedblood pressure. Reperfusion (90 min) was confirmed by the lack of cy-anosis in that region. Exclusion criteria were dysrhythmia and/ora sustained fall in mean arterial blood pressure below 60 mmHgbefore occlusion.

Infarct Size Measurement

At the end of 90 min reperfusion, the LAD was reoccluded and 2 mLEvans blue (1%) injected as a bolus into the tail vein and allowed toperfuse the nonischemic portion of the heart as the blue area. The en-tire heart was then excised, rinsed of excess blue dye, trimmed of

atrial tissue and frozen at –20 �C. The hearts were then sliced into2 mm thick transverse sections from the apex to the base. The ische-mic area or area at risk (AAR) was cut down and incubated in 1% tri-phenyltetrazolium chloride (TTC) solution in isotonic pH 7.4phosphate buffer at 37 �C for 20 min. Samples were fixed in 10% for-malin for 24 h to enhance the contrast. An image was obtained fromboth sides of every slice and all calculations from one heart (using Im-age Tool Software, San Antonio, TX) were averaged into one value forstatistical analysis. Infarct size, as the white color area, was ex-pressed as a percentage of AAR (as the red color area).

Measurement of Plasma Creatine Kinase (CK) Activity

At the end of reperfusion, the blood samples (2 mL) were collectedfrom carotid artery. Plasma was obtained by centrifugation at2500 g for 15 min (4 �C). Plasma CK activity was determined by stan-dard kits (Parsazmoon, Tehran, Iran) using an autoanalyzer and ex-pressed as unit per milliliter (U/mL).

Ischemic-Induced Arrhythmia Analysis

Arrhythmia were defined according to the Lambeth conventions[22] in which ventricular premature beats (VEBs) were defined as dis-crete and identifiable premature QRS complex and ventricular tachy-cardia (VT) as a run of four or more consecutive VEBs. Ventricularfibrillation was defined as a signal where individual QRS deflectioncould not easily be distinguished from each other and where heartrate could no longer be measured. Complex forms (bigeminy and sal-vos) were added to VEBs count and not analyzed separately.

Experimental Protocol

In our previous study [19], it was observed that 120 min of exposureto normobaric hyperoxia 95% is the optimum time and concentrationto induce late hyperoxic preconditioning. These time and oxygen con-centrations were selected for the present study as follow:

Group NOR: normoxia (21% O2 for 120 min) 24 h before ischemia(n¼ 14)Group H12: hyperoxia (�95% O2 for 120 min) 12 h before ischemia(n¼ 11)Group H24: Hyperoxia (�95% O2 for 120 min) 24 h before ischemia(n¼ 12)Group H48: hyperoxia (�95% O2 for 120 min) 48 h before ischemia(n¼ 9)Group H72 : Hyperoxia (�95% O2 for 120 min) 72 h before ischemia(n¼ 12)

Rats in hyperoxic groups were exposed to normobaric hyperoxia(O2� 95%) for 120 min in an air-tight chamber with a small inletand outlet. The chamber was continuously ventilated with nearlypure oxygen. The O2 concentration of chamber was continuously mon-itored with an oxygenmeter (Lutron Do5510; Taiwan). To preventCO2 retention, soda lime was placed in the chamber. All rats werethen allowed to breathe normal air for 12, 24, 48, and 72 h before theirhearts were subjected to 30 min ischemia and 90 min reperfusion. Thenormoxic group was placed in the same container for 120 min, with aninflow of normal air.

Second series of experiments were designed to investigate whetherintermittent hyperoxia preconditioning (pre-exposure to the samenormobaric hyperoxia 24 h prior to the end of the late phase of thelast preconditioning in which the infarct size was assumed as a goldstandard) could potentate and prolong the protective effects of hyper-oxia preconditioning. In these series, each group was chosen accord-ing to the results of previous group as follows:

Group NOR; rats were exposed to normoxia for 120 min once per d,24 h before being subjected to I/R (n¼ 14)

M(HAnan

Gr

FirNOH1H2H4H1SecH2H2H3H3

Nexpreswedifrep

BAHARVAND ET AL: PRECONDITIONING AND INTERMITTENT HYPEROXIA 55

Group H48; rats were exposed to hyperoxia for 120 min once per d,48 h before being subjected to I/R (n¼ 9)Group H2-48; rats were exposed to hyperoxia for 120 min once perd for 2 consecutive d, 48 h before being subjected to I/R (n¼ 8)Group H2-72; rats were exposed to hyperoxia for 120 min once perd for 2 consecutive d, 72 h before being subjected to I/R (n¼ 7)Group H3-73; rats were exposed to hyperoxia for 120 min once perd for 3 consecutive d, 72 h before subjected to I/R (n¼ 7)Group H3-96; rats were exposed to hyperoxia for 120 min once perd for 3 consecutive d, 96 h before being subjected to I/R (n¼ 7)

Statistical Analysis

Data are expressed as mean 6 SEM and the percentage of inci-dence. Statistical comparison of means between groups was madeby one-way ANOVA followed by Tukey’s post-hoc test. Within eachgroup, the data of homodynamic parameters were compared usingone-way repeated measures ANOVA. The incidences of VF and sur-vival rate were compared using the Fisher exact test. P< 0.05 wasconsidered to be statistically significant.

RESULTS

First series of Experiments

Hemodynamic ParametersThe hemodynamic parameters are summarized in

Table 1. There was no significant difference at baselinevalues for heart rate and mean arterial blood pressureamong all the groups. During ischemic period, theonly factor that reduced significantly was the arterialblood pressure in all the groups, which returnedapproximately to normal values.

TABLE 1

ean Arterial Blood Pressure (MBP) and Heart RateR) Before and During Ischemia and Reperfusion inesthetized Rats Exposed to Normoxia, Hyperoxia,d Intermittent Hyperoxia

Baseline Ischemia Reperfusion

oups HR MBP HR MBP HR MBP

st series experimentsR 360 6 12 93 6 4.9 364 6 9.9 71 6 4.1** 355 6 8.5 87 6 3.32 359 6 11 95 6 3.5 366 6 13 78 6 2.9* 362 6 10 93 6 4.84 383 6 9 89 6 4.2 376 6 8 70 6 3.4 * 360 6 9 80 6 3.78 363 6 9 89 6 4.3 374 6 9 64 6 3.4** 355 6 14 82 6 4.472 383 6 13 92 6 4.8 353 6 11 74 6 3.6** 366 6 12 89 6 5.3ond series experiments-48 354 6 12 91 6 3.8 361 6 8 76 6 4.7* 366 6 11 83 6 3.3-72 342 6 8 93 6 4.5 351 6 7 74 6 6.1** 371 6 13 92 6 3.9-72 368 6 10 89 6 4.3 382 6 12 70 6 5.2** 361 6 10 91 6 6.1-96 351 6 14 97 6 5.1 360 6 10 76 6 4.9* 358 6 12 92 6 4.2

OR: normoxic groups; H12, H24, H48, and H72 referring to ratsosed to hyperoxic air (O2> 95%) 12, 24, 48, and 72 h later,pectively. H48, H2-48, H2-72, H3-72, and H3-96 referring to ratsre pre-exposed to hyperoxia for 1, 2, or 3 d (120 min once a d) atferent times before a 30-min ischemia followed by a 90-minerfusion.*P< 0.05.**P< 0.01 compared with NOR.

Infarct Size and Plasma CK ActivityFigure 1A represents infarct size as a percentage of

area at risk. There was no significant difference in thearea at risk among all the groups (data not shown).The infarct size was 48.1% 6 4% and 44.2% 6 4.2% incontrol and H12 groups, respectively. It was signifi-cantly reduced in H24 (31.3% 6 3.3%) and H48(27.7% 6 2.9%) groups. There was no marked differenceamong the NOR and H72 (41.8% 6 3.6%) groups. Theplasma CK activity was in consistent to infarct size(Fig. 1B). It was reduced significantly in H24 and H48groups. Plasma CK activity was 1.52 6 0.06,1.55 6 0.05, 1.03 6 0.05, 1.26 6 0.04, and 1.54 6 0.06U/mL in NOR, H12, H24, H48, and H72 groups, respec-tively.

Ischemic I induced ArrhythmiaThe episodes of VEBs decreased from 340 6 35 and

293 6 38 in NOR and H12 groups, to 173 6 20 and190 6 23 in H24 and H48 groups, respectively. The dif-ference between NOR and H72 (274 6 36) groups was

FIG. 1. The effect of pre-exposure to normobaric hyperoxia(O2> 95%, 120 min) at different times before 30-min ischemia and90 min-reperfusion on infarct size (A) and plasma CK activity (B) inopen chest heart rats. NOR: Normoxic groups; H12, H24, H48, andH72 rats exposed to hyperoxic air (O2> 95%) 12, 24, 48, and 72 hlater, respectively. *P< 0.05 and **P< 0.01 compared with NORgroup.

JOURNAL OF SURGICAL RESEARCH: VOL. 160, NO. 1, MAY 1, 201056

not significant (Fig. 2A). The episodes of VT only re-duced in H24 group. It was 41.3 6 5.3, 42.4 6 6.6,18.8 6 3.8, 30.2 6 5.1, and 39 6 7.7 in NOR, H12, H24,H48, and H72 groups, respectively (Fig. 2B). The inci-dences of VF were 66.6% in NOR group, 85% in H12group, and 62.5% in H72 group without any signifi-cance, whereas, it had a marked reduction in H24(30%) and H48 (42.8 %) groups (Fig. 2C).

Survival RateThe survival rate was 58% in NOR and 63 % in H12

groups. It was increased to 92 % in H24 and to 77.7%in H48 group. There was no difference between NORand H72 (77.7 %) groups (Fig. 2D).

Second Series of Experiments

In these experiments we hypothesized that the inter-mittent pre-exposure to hyperoxia could potentiate andprolong the late phase of hyperoxia preconditioning. Inorder to examine this hypothesis, we modified theparameters according to the data obtained from thefirst series of experiments. For instance, we did not ob-served anti-infarct effect 72 h after exposures for 2 con-secutive d, so the time of exposure was changed from 2d to 3 d.

FIG. 2. The effect of pre-exposure to normobaric hyperoxia (O2> 95perfusion on ischemic-induced arrhythmia in open chest heart rats. VEBventricular fibrillation; NOR: normoxic groups; H12, H24, H48, and H7respectively. *P< 0.05, **P< 0.01, and ***P< 0.001 compared with NO

Hemodynamic ParametersTable 1 shows the heart rate and mean arterial blood

pressure at the baseline values among all the groups.During ischemic period, the only factor that reduced sig-nificantly was the arterial blood pressure in all thegroups, which returned approximately to normal values.

Infarct SizeAs Fig. 3A shows, there was a significant difference

between the infarct size of H48 (27.7% 6 2.9%) andH2-48 (24.2% 6 2.5%) compared with NOR group.There was no marked difference between H48 andH2-48 groups. The infarct size was 36.7% in H72 group.When the pre-exposure times were increased to 3 con-secutive d (H3-72 group), the infarct size reduced signif-icantly 72 h later (28.5% 6 3.6%), but not 96 h later(H3-96 group; 46.1% 6 4%). The plasma CK activityhad a similar pattern with the changes in the infarctsize (Fig. 3B). It was 1.52 6 0.06, 1.26 6 0.0,1.13 6 0.07, 1.37 6 0.07, 1.13 6 0.06, and 1.44 6 0.08U/ mL in NOR, H48, H2-48, H2-72, H3-72, and H3-96groups, respectively.

Ischemic-Induced ArrhythmiaFigure 4A–C show the episodes of VEB and VT and

the incidence of VF during ischemic period. The

%, 120 min) at different times before 30 min-ischemia and 90 min-re-: ventricular ectopic beat; VTe: episodes of ventricular tachycardia; VF:2, rats exposed to hyperoxic air (O2> 95%) 12, 24, 48, and 72 h later,R.

FIG. 3. The effect of pre-exposure to intermittent normobaric hy-peroxia (O2> 95%, 120 min once a d) at different times before 30-minischemia and 90-min reperfusion on infarct size (A) and plasma CKactivity (B) in open chest heart rats. NOR: normoxia; H48, H2-48,H2-72, H3-72, H3-96 rats were pre-exposed for 1, 2, or 3 d (120 minonce a d) at different times before 30 min ischemia and 90 min reper-fusion. *P< 0.05, **P< 0.01, and ***P< 0.001 compared to NOR.

BAHARVAND ET AL: PRECONDITIONING AND INTERMITTENT HYPEROXIA 57

episodes of VEBs decreased from 314 6 25 in NORgroup to 190 6 22, 183 6 25, and 167 6 22 in H48, H2-48, H3-72 groups, respectively (Fig. 4A). There was nomarked difference between NOR and H2-72(252 6 22) and H3-96 (279 6 32) groups.

Although there was no marked significant differ-ences between all the groups in VT episodes, however,the pattern of changes was similar to that of VEBepisodes (Fig. 4B). It was 41.3 5.3, 30.25.1, 21.52.9,30.55.8, 23.44.6 and 35.74.8 in NOR, H48, H2-48, H2-72, H3-72 and H3-96, respectively. Figure 4C demon-strates that VF% decreased significantly in all thegroups except H3-96 group. It was 66.6, 42.8, 28, 42.8,14.2, and 57.1 % in NOR, H48, H2-48, H2-72, H3-72,and H3-96 groups, respectively. VF% had a more reduc-tion in H2-48 and H3-72 groups than H48 group.

Survival RateSurvival rate was 58% in NOR group and it was in-

creased in all the groups considerably compared with

NOR group, except H3-96 group (Fig. 4D). It was 58,77.7, 88, 78.8, 100, and 64% in NOR, H48, H2-48, H2-72, H3-72, and H3-96 groups, respectively. The survivalrate had a significant increase in H2-48 and H3-72groups than H48 group.

DISCUSSION

Our salient findings of this study were: (1) exposingrats to normobaric hyperoxia for at least 120 min mayprotect their heart against ischemic-induced arrhyth-mia and infarction, which lasts for approximately48 h; (2) intermittent pre-exposure to the same hyper-oxic environment for at least 3 consecutive d (120 min,once a d), could prolong this protection. These protec-tions were consistent with plasma CK activity. Increas-ing the protection of tissues against a prolongedischemic injury has a great interest in clinical realm.Since Murry et al. in 1986 [23], it has been reportedthat preconditioning by sublethal stimulus induces a bi-phasic tolerance against a subsequent lethal insult:with an early phase that lasts for 2 to 3 h and a delayedphase that initiates about 24 h later and continues up to72 h [19, 24]. Several studies using infusion of low con-centrations of oxygen radicals [3, 4, 6, 25] and adminis-tration of antioxidants [26–28] have demonstrated thatROS acts as the trigger of preconditioning process. Re-cent studies have shown that pre-exposure to normo-baric hyperoxia, as a low graded systemic oxidativestress, could induce preconditioning like-effects in ratand mouse hearts [9, 11, 29–32]. In our previous study,we demonstrated that pre-exposure to normobaric hy-peroxia could enhance the tolerance of myocardiumagainst ischemic induced arrhythmia and infarction24 h later in anesthetized rats [19]. The results wereconsistent with the report of Tahepold’s group using iso-lated rat and mouse heart [20, 21]. In this study, weshowed that these protective effects of hyperoxia pre-treatment last for about 48 h in anesthetized rats. Thereis no study about the length of the late phase of normo-baric hyperoxia in different organs, but our study wassimilar to the study of Choi’s group using hyperbaric hy-peroxia in the isolated rat hearts [10].

As it was noted in the previous section, the delayedphase of preconditioning lasts for up to 72 h [10], so po-tentiating and prolonging the late phase of hyperoxiapreconditioning might cause a great interest in clinicalmedicine. To our knowledge, there is no such studyabout prolonging the late phase of hyperoxia-inducedpreconditioning. Recently, Kaljusto and co-workershave indicated that the combination of the late phaseof dexamethasone and the early phase of hyperoxia pre-conditioning improved postischemic heart function,however, did not reduce infarct size compared with

FIG. 4. The effect of pre-exposure to intermittent normobaric hyperoxia (O2> 95%, 120 min once a d) at different times before 30 minischemia and 90 min reperfusion on ischemic-induced arrhythmia in open chest heart rats. NOR: normoxia; H48, H2-48, H2-72, H3-72, H3-96 refers to rats pre-exposed for 1, 2, or 3 d (120 min once a d) at different times before 30 min ischemia and 90 min reperfusion. *P< 0.05,**P< 0.01, and ***P< 0.01 compared with NOR and , P< 0.01 and b, P< 0.001 versus H48 group.

JOURNAL OF SURGICAL RESEARCH: VOL. 160, NO. 1, MAY 1, 201058

the single pretreatment [33]. In the second series of ourexperiments when rats were exposed to hyperoxic envi-ronment once a d for 2 consecutive d, the incidence ofVF decreased and survival rate increased remarkably,72 h later. But, the infarct size did not decrease(Fig. 4). When the numbers of exposures were increasedto 3 d, both the infarct size and arrhythmia reduced72 h later. These results indicate that prolonging theprotection against infarction or arrhythmia by pre-treatment with intermittent hyperoxia depends on thenumber of exposure.

Pre-exposure to hyperoxia could induce precondition-ing effect in other organs, however pre-exposure timesare very different for different organs [14, 15, 32, 34].Most of these studies have used intermittent hyperoxiato induce preconditioning. Our colleagues observed hy-peroxia preconditioning effect in rat kidneys when theywere exposed to hyperoxic environment at least for 1 honce a d for 5 consecutive d [18]. Zhang et al. observedneuroprotective effect with 24 h consecutive exposureto hyperoxia, but did not observe the same effect with6 and 12 h of exposure [35]. So, we hope that next stud-ies using the different methods of hyperoxia could finda more optimal time and concentration of oxygen thatprecondition all organs over the body.

In summary, although prolonged exposure to hyper-oxia (more than 24 h) has detrimental effects [8, 36],transient exposure may induce a late preconditioningthat lasts for about 48 h, and intermittent pre-exposure

to the same hyperoxia might prolong this protection inthe hears of rats.

ACKNOWLEDGMENTS

This work was supported by a grant from Trauma Research Centerof Baqiyatallah University of Medical Sciences.

The authors thank Professor Alireza Asgari for critical review onthe manuscript.

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