Kaohsiung Journal of Medical Sciences (2013) 29, 119e127

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ORIGINAL ARTICLE

Postconditioning attenuates acute intestinalischemiaereperfusion injury

Ilker Sengul a,*, Demet Sengul b, Osman Guler c, Adnan Hasanoglu c,Mustafa Kemal Urhan c, Ahmet Sukru Taner c, Jakob Vinten-Johansen d

aDepartment of General Surgery, Giresun University Faculty of Medicine, Giresun, TurkeybDepartment of Pathology, Giresun University Faculty of Medicine, Giresun, TurkeycDepartment of 1st General Surgery, Ankara Education and Research Hospital, Ankara, TurkeydDepartment of Surgery (Cardiothoracic), Cardiothoracic Research Laboratory of Emory University, CarlyleFraser Heart Center, Atlanta, GA, USA

Received 12 October 2011; accepted 22 February 2012Available online 10 October 2012

KEYWORDSCreatine kinase;Intestinal ischemiaereperfusion;Ischemiaereperfusioninjury;Malondialdehyde;Postconditioning

* Corresponding author. Giresun UnNizamiye Yerleskesi, 28100 Giresun, T

E-mail addresses: dr.ilker52@myne

1607-551X/$36 Copyright ª 2013, Kaohttp://dx.doi.org/10.1016/j.kjms.201

Abstract The aim of this study was to test the hypothesis that postconditioning (POC) wouldreduce the detrimental effects of the acute intestinal ischemiaereperfusion (I/R) compared tothose of the abrupt onset of reperfusion. POC has a protective effect on intestinal I/R injury byinhibiting events in the early minutes of reperfusion in rats. Twenty-four WistareAlbino ratswere subjected to the occlusion of superior mesenteric artery for 30 minutes, then reperfusedfor 120 minutes, and randomized to the four different modalities of POC: (1) control (no inter-vention); (2) POC-3 (three cycles of 10 seconds of reperfusionereocclusion, 1 minute totalintervention); (3) POC-6 (six cycles of 10 seconds of reperfusionereocclusion, 2 minutes totalintervention); and (4) sham operation (laparotomy only). The arterial blood samples [0.3 mLtotal creatine kinase (CK) and 0.6 mL malondialdehyde (MDA)] and the intestinal mucosalMDA were collected from each after reperfusion. POC, especially POC-6, was effective inattenuating postischemic pathology by decreasing the intestinal tissue MDA levels, serum totalCK activity, inflammation, and total histopathological injury scores. POC exerted a protectiveeffect on the intestinal mucosa by reducing the mesenteric oxidant generation, lipid peroxida-tion, and neutrophil accumulation. The six-cycle algorithm demonstrated the best protection.Copyright ª 2013, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. All rightsreserved.

iversitesi Tip Fakultesi Dekanligi, Dekan Yardimcisi, Genel Cerrahi Anabilim Dali Kurucu Baskani,urkey.t.com, ilker.sengul@giresun.edu.tr (I. Sengul).

hsiung Medical University. Published by Elsevier Taiwan LLC. All rights reserved.2.08.021

Control 30 min I

POC-330min I R I R I R I

POC-6 30 minI R I R I R I R I R I R I

Sham

Each cycle of 10 s R/I

2 h R

2 h R

2 h R

Figure 1. A schematic diagram showing the POC protocols inthe various groups. I Z ischemia; POC Z postconditioning;POC-3 Z three cycles of postconditioning; POC-6 Z six cyclesof postconditioning; R Z reperfusion.

120 I. Sengul et al.

Introduction

Murry and colleagues [1] introduced the concept ofischemic preconditioning (IPC) in which repetitive briefepisodes of ischemia render the myocardium more resistantto a subsequent prolonged ischemic insult that causesirreversible injury. In recent years, the VinteneJohansenLaboratory reported in the canine coronary artery occlu-sionereperfusion model that a similar regimen of briefepisodes of ischemia carried out just after, instead of justbefore, the prolonged (index) ischemia reduced infarctsize, coronary artery endothelial dysfunction, and neutro-phil accumulation in the area at risk. This strategy, termedpostconditioning (POC), was subsequently found to reduceinfarct size comparably to that of IPC. While POC wasinitially studied in the heart [2], it has also been reportedto protect the tissue following ischemia in the liver [3],brain [4], kidney [5], skeletal muscle [6], and skin flaps [7].Ferencz and colleagues [8] reported that POC decreasedinjury to the intestinal mucosa in the intestinal grafts after6 hours of preservation with University of Wisconsin solu-tion. However, studies on the intestinal protection exertedby POC after in situ ischemia are limited [9].

The efficacy of POC as a reperfusion therapy was basedon the underlying hypothesis that significant reperfusionpathology occurs during the early minutes of reperfusion.Studies by Yang et al. [10] and Kin et al. [11] suggested thatendogenous mechanisms are set in motion within the firstfew minutes of reperfusion that attenuate the earlycomponents of reperfusion injury. Early events of reperfu-sion injury are not independent of one another, but form anarray that also triggers later events such as increasedcapillary permeability, no reflow, apoptosis, and necrosis.

Although all the protective mechanisms of POC are stillnot known, those that have been identified include delayedopening of the mitochondrial permeability transition pore(mPTP) [12], activation of mitochondrial adenosine triphos-phate (ATP)-sensitive potassium (mKATP) channels [13],prevention of mitochondrial peroxide production and gluta-thione depletion [14], activation of components of thereperfusion injury salvage kinase pathway [15,16], activa-tion of the nitric oxideeguanylate cyclase and protein kinaseG pathway [17], and inhibition of reactive oxygen speciesproduction and intracellular excessive calciumaccumulation[18]. Of these many targets, attenuation of reactive oxygenspeciesmay be amajormechanism because reduced oxidantgeneration would attenuate direct tissue injury as well asremove amajor stimulus that opens themPTPat reperfusion.

Although POC has been demonstrated to protect manyorgan types, the optimal algorithm is not known. Somestudies suggest that the number of POC cycles is moreimportant than their duration, but this is controversial(reviewed in Ref. [19]). The question of the optimal algo-rithm has not been investigated in the intestinal tractundergoing ischemia and reperfusion. Specifically, it is notknown whether increasing the number of cycles increasesthe degree of protection in the intestinal tract. In thecurrent study, we tested the hypothesis that increasing thenumber of POC cycles after the intestinal ische-miaereperfusion (I/R) against the ischemic injury in the ratwould reduce postischemic injury.

Materials and methods

Animal care

All the animals received humane care in compliance with“The Guide for the Care of Use of Laboratory Animals”published by the National Institute of Health (NIH Publica-tion No. 85-23, revised 1996), as well as with Turkish lawson animal experimentation. The present study received theprevious approval of the ethics committee of AnkaraEducation and Research Hospital.

Surgical preparation

Twenty-four male and female WistareAlbino rats weighing200e250 g were obtained from the Animal Laboratory ofAnkara Education and Research Hospital. The male andfemale rats were equally distributed among the experi-mental groups. The animals were anesthetized with anintramuscular injection of ketamine (50 mg/kg) and xyla-zine (7 mg/kg), and ventilated with the room air. Theagents S-ketamine and xylazine were chosen because theydo not exert a preconditioning-like effect by activation ofthe mKATP channel [20]. The rats were placed in a supineposition on a heating pad to maintain the body temperaturebetween 37�C and 38�C. To induce intestinal ischemia,a laparotomy was performed under the sterile conditions,and the superior mesenteric artery was occluded with anatraumatic arterial clamp. The abdominal operative zonewas covered with a sterile saline-soaked gauze and a plasticcover to minimize dehydration of exposed tissues duringthe experiment.

Experimental protocol

In all rats, the proximal superior mesenteric artery wasoccluded for 30 minutes and then reperfused by looseningthe atraumatic arterial clamp for 120 minutes. The ratswere randomly assigned to one of four groups based on theintervention (n Z 6 in each group) (Fig. 1): (1) con-troldthere was no intervention either prior to or aftersuperior mesenteric artery occlusion; (2) POC-3dimmediately at the onset of reperfusion, reflow wasinitiated with 10 seconds of full mesenteric flow (reperfu-sion), followed by 10 seconds of reocclusion (ischemia),repeated for a total of three cycles (1 minute total inter-vention); (3) POC-6dthe refloweocclusion perioddescribed above was repeated for six cycles (2 minutes

Intestinal postconditioning 121

total intervention); and (4) shamdlaparotomy only withthe observation time equal to the total experimental time.

Determination of serum total creatine kinase andserum malondialdehyde activity

The arterial blood samples [0.3 mL for total creatine kinase(CK) and 0.6 mL for malondialdehyde (MDA)] were collectedfrom each rat after 120 minutes of reperfusion. Plasma wasanalyzed spectrophotometrically for CK activity at 350 nmabsorbance. To measure plasma MDA levels, an index oflipid peroxidation reflecting oxygen free radicals, themethod of Hunter et al. [21], was used. The MDA producthas a long half-life in the plasma, and therefore its plasmalevels are cumulative over time. The plasma was stored at�70�C until analyzed. CK activity was expressed as unitsper liter of the plasma and MDA values as nanomoles permilliliter of the plasma.

Determination of intestinal mucosal MDA

TheUchiyamamethod [22] was used to determine theMDA inthe intestinal mucosa. The weight of each sample of intes-tinal mucosa was measured. The tissue samples werehomogenized and cold 1.5% potassium chloride (KCl) wasadded to achieve a 10% homogenate. Then 3 mL of 1% phos-phoric acid and 1 mL of 0.6% thiobarbituric acid were addedto 0.5 mL of the homogenate. After boiling and refrigeratingfor 45 minutes, 4 mL of N-butanol were added, and thesamples were centrifuged at 3500� for 10 minutes to sepa-rate butanol gas. Absorbances of these gases were analyzedspectrophotometrically at 520 and 535 nm, and the differ-ences of absorbances were measured as the MDA levels.

Histopathologic assessment

The postischemic intestinal tissue samples were rinsedpromptly in a cold saline and immediately fixed for 48 hours

Table 1 System of histopathological scores of Chiu et al. [23] (

Score

Mucosal injury 012345

Inflammation 012345

Hyperemia/hemorrhage 012345

in a 10% buffered formalin. The tissues were thenembedded in paraffin and sectioned transversely followingroutine procedures. Serial sections were stained withhematoxylin and eosin, and evaluated by the light micros-copy. Photomicrographs were taken at 10�/0.25 and 40�/0.65 magnification by two separate investigators blinded tothe previous interventions. Morphological evaluationwas performed according to the Chiu classification [23](Table 1). [23] The intestinal mucosal injury in each slidewas graded on a six-tiered scale as follows: grade0 Z normal mucosa; grade 1 Z development of sub-epithelial (Gruenhagen’s) spaces near the tips of villi withthe capillary congestion; grade 2 Z extension of the sub-epithelial spaces with moderate epithelial lifting from thelamina propria; grade 3 Z significant epithelial lifting alongthe length of the villi with a few denuded villus tips; grade4 Z denuded villi with exposed lamina propria and dilatedcapillaries; and grade 5 Z disintegration of the laminapropria, with hemorrhage and ulceration.

Statistical analysis

All data were expressed as means � standard error ofmeans. Differences between the groups were analyzed byKruskaleWallis one-way analysis of variance. All the datawere analyzed using SPSS for Windows 11.5 and p < 0.05was considered significant.

Results

Plasma total CK activity

The total plasma CK activity after I/R (as shown in Fig. 2)was significantly greater in the control group than in thesham group (p < 0.05, p Z 0.003). In the POC-3 group, theplasma CK activity did not alter significantly relative to thatin the control group. However, plasma CK activity in POC-6group was significantly reduced relative to the control and

Chiu classification).

Determination

Normal mucosaSubepithelial gaps of villiModerate separation of epitheliumIntensive separation of epitheliumEvanescence of villi and manifestation of the lamina propriaBreaking into pieces of lamina propria with ulcerationNoneIn lamina propria, locallyIn lamina propria, diffuselyLocal subendothelial collectionsDiffuse subendothelial collectionsMassive collectionsNoneDilated capillaries in lamina propriaLocal hemorrhage in lamina propriaDiffuse hemorrhage in lamina propriaSubendothelial hemorrhageMassive hemorrhage

Figure 2. A comparative graph of the plasma total CKactivities (U/L plasma) of the groups (p < 0.05, p Z 0.003).The values are group means � SE. CK Z creatine kinase;POC-3 Z three cycles of postconditioning; POC-6 Z six cyclesof postconditioning; SE Z standard error.

122 I. Sengul et al.

POC-3 groups, but was comparable to that in the shamgroup. Therefore, the six-cycle algorithm of POC reducedthis biomarker of the morphological tissue injury.

Plasma MDA levels

Changes in the plasma MDA levels are summarized inFig. 3A. The plasma MDA levels were significantly greater inthe control group subjected to I/R compared to that in thesham group (p < 0.05, pZ 0.012). The plasma MDA levels inboth POC groups tended to be lower than in the controlgroup, but this was not a significant difference.

Figure 3. (A) A comparative graph of the plasma MDA levelsof the groups (p < 0.05, p Z 0.012). Values are groupmeans � SE. (B) A graph of the intestinal tissue MDA levels inthe experimental groups. The values are group means � SE.MDA Z malondialdehyde; POC-3 Z three cycles of post-conditioning; POC-6 Z six cycles of postconditioning;SE Z standard error.

Intestinal tissue MDA levels

As shown in Fig. 3B, the intestinal tissue MDA levels weresignificantly elevated in the control group compared to thatin the sham group. POC-3 group was not associated witha significant reduction in tissue MDA, but in the POC-6 groupthe tissue MDA was significantly reduced compared to bothcontrol and POC-3 groups.

Postexperimental histopathological assessment

Assessment of each slide was performed according to theChiu classification and differences between the groupswere evaluated using the parameters of mucosal injury,inflammation, and hyperemia/hemorrhage (Table 1).

Mucosal injury scores

As shown in Fig. 4A, [23] I/R injury was associated withgreater mucosal injury scores in the control group than inthe sham group. The mucosal injury score was not signifi-cantly reduced in the POC-3 group compared to that in thecontrol group. However, the POC-6 group was associatedwith a significantly lower mucosal injury score compared tothe control and POC-3 groups. Fig. 4BeE show the photo-micrographs representative of the different scores for thePOC-6, control, and sham groups.

Inflammation scores

As shown in Fig. 5A, [23] the inflammation scores in thecontrol group were significantly greater compared to allother interventional groups (p < 0.05, p Z 0.000). Inflam-matory scores were significantly lower in both POC-3 andPOC-6 groups than in the control group. Fig. 5BeE show thephotomicrographs representative of the different scores forthe POC-3, POC-6, and sham groups.

Hyperemia/hemorrhage scores

There was no significant difference in the tissue hyper-emia/hemorrhage scores among all experimental groups(Fig. 6A). [23] Fig. 6BeD show the photomicrographsrepresentative of the different scores for the POC-6, POC-3, and sham groups.

Total scores

As shown in Fig. 7, [23] the average total score in thecontrol group was significantly greater compared to allother interventional groups (p < 0.05, p Z 0.004). BothPOC-3 and POC-6 algorithms reduced the total injury scoresrelative to control. There was no significant differencebetween the POC-3 and POC-6 groups.

Discussion

I/R injury is a contributing factor to mortality and morbidityfrom myocardial infarction, septic shock, and multiorganfailure (MOF). It has been reported that intestinal

Figure 4. (A) A comparative graph of the histopathological analysis of mucosal injury scores of rats in the groups according toclassification of Chiu et al. [23]. (B) Note the subepithelial gaps on the top of mucosal villi (indicated by arrow) (mucosal injury,score 1) in the POC-6 group (hematoxylin and eosin stain; original magnification, 40�). (C) Note the intensive separation of theepithelium (indicated by arrow) (mucosal injury, score 3) in the control group (hematoxylin and eosin stain; original magnification,20�). (D) Note the evanescence villi and manifestation of the lamina propria (indicated by arrow) (mucosal injury, score 4) in thecontrol group (hematoxylin and eosin stain; original magnification, 20�). (E) Note the normal mucosa (mucosal injury, score 0) inthe sham group (hematoxylin and eosin stain; original magnification, 5�). POC-6 Z six cycles of postconditioning; SE Z standarderror.

Intestinal postconditioning 123

hypoperfusion triggers a systemic inflammatory responsethat leads to MOF [24]. The gastrointestinal tissue is highlysensitive to I/R injury. However, in addition to affecting thegastrointestinal tissue, this injury causes a secondary injuryto the remote tissue such as the lung. The gastrointestinaltissue is the target of the oxidant injury and is itselfa source of inflammation as enterocytes can generate theproinflammatory cytokines that may lead to enterocytedysfunction, bacterial translocation [25], and the largerproblem of sepsis and its MOF [26]. Hence, protection ofgastrointestinal tract from I/R injury is important. IPC hasbeen reported to reduce postischemic gastrointestinalinjury [9,27]. However, it has limited applicability inprotection of the gastrointestinal tract as it does in the

heart [9] and other organs. However, recently POC has alsobeen reported to attenuate postischemic gastrointestinalinjury [8,28e33]. Contrarily, Bretz et al. [34] propoundedthat POC was ineffective in decreasing I/R injury of thesmall intestines in a rabbit model of intestinal I/R. For allthat, Ferencz et al. [8] reported that POC alleviate oxida-tive stress and histologic damage of the small intestinesduring the small bowel autotransplantation. They per-formed total orthotopic intestinal autotransplantation in 30white domestic pigs and stored the grafts in cold Universityof Wisconsin solution for 1, 3, or 6 hours. They found thatthe group of POC significantly decreased the reperfusion-ended lipid peroxidation, endogenous antioxidant protec-tive systems (glutathione and superoxide dismutase), and

Figure 5. (A) A comparative graph of the histopathological analysis of inflammation scores of rats in the groups according toclassification of Chiu et al. [23] (p < 0.05, p Z 0.000). Values are group means � SE. (B) Note the diffuse subendothelial collections(indicated by three arrows) (inflammation, score 4) in the POC-3 group (hematoxylin and eosin stain; original magnification, 20�).(C) Note the massive collections (indicated by arrow) (inflammation, score 5) in the control group (hematoxylin and eosin stain;original magnification, 20�). (D) Note the inflammation in the lamina propria, locally (indicated by arrow) (inflammation, score 1),in the POC-6 group (hematoxylin and eosin stain; original magnification, 20�). (E) A photomicrograph showing no inflammation(inflammation, score 0) in the sham group (hematoxylin and eosin stain; original magnification, 20�). POC-3 Z three cycles ofpostconditioning; POC-6 Z six cycles of postconditioning; SE Z standard error.

124 I. Sengul et al.

intestinal wall injury. Additionally, it was detected that thetissue injury was increased by the duration of cold preser-vation (mostly in 6 hours of preservation). They performedthe three cycles of ischemia for 30 seconds and reperfusionfor 30 seconds.

In the present study, we report the following occur-rences: (1) mesenteric I/R was associated with significantinjury to the mucosa and submucosal tissues; (2) POCreduced the postischemic injury by attenuating themembrane lipid products of oxidant injury and inflamma-tory processes; (3) the three-cycle algorithm was largelyineffective, but increasing the number of POC cycles to six

was effective in reducing postischemic injury. Thesestudies suggest that the postischemic mesenteric injury canbe reduced by six cycles of POC applied at the time ofreperfusion, consistent with the reduction of early post-ischemic events reported for the heart [2,19,35].

The tissue-protective effect of POC is exerted by thealternating “units” of reperfusion and reocclusion(ischemia). There is controversy over whether the efficacyof POC is due primarily to the number of cycles, duration ofeach (reperfusionereocclusion) period, or total duration ofthe POC algorithm. However, it is clear that each of thealternating phases contributes to the cardioprotection of

Figure 6. (A) A comparative graph of the histopathological analysis of hyperemia and hemorrhage scores of rats in the groupsaccording to the classification of Chui et al. [23]. Values are group means � SE. (B) Note the dilated capillaries in the lamina propria(indicated by arrow) (hyperemia and hemorrhage, score 1) in the POC-6 group (hematoxylin and eosin stain; original magnification,20�). (C) Note the diffuse hemorrhage in the lamina propria (indicated by two arrows) (hyperemia and hemorrhage, score 3) in thePOC-3 group (hematoxylin and eosin stain; original magnification, 20�). (D) Note the subendothelial hemorrhage in the laminapropria (indicated by three arrows) (hyperemia and hemorrhage, score 4) in the control group (hematoxylin and eosin stain;original magnification, 40�). POC-3 Z three cycles of postconditioning; POC-6 Z six cycles of postconditioning; SE Z standarderror.

Intestinal postconditioning 125

POC. For example, the ischemic phase maintains tissueacidosis [36] part through inhibition of calpain activation[37] and in part by inhibiting the opening of the mPTP [36],the latter event being a potential final common pathway ofthe tissue protection. Kin et al. [38] reported in a rat modelof coronary artery reperfusion that the three cycles ofreperfusioneocclusion were as effective as six cycles. Yanget al. [10] reported a similar degree of the protection with

Figure 7. A comparative graph of the histopathologicalanalysis of total scores of rats in the groups according toclassification of Chui et al. [23] (p < 0.05, p Z 0.004). Thevalues are group means � SE. POC-3 Z three cycles of post-conditioning; POC-6 Z six cycles of postconditioning;SE Z standard error.

four and six cycles of reperfusioneocclusion. However, theeffective algorithm has not been explored in other tissues,including the mesentery. In the present study, the six-cyclealgorithm was more protective than the three-cyclealgorithm.

POC inhibits a number of the pathophysiological mech-anisms leading to cell injury. The data suggest that inhibi-tion of the inflammatory response to the reperfusion is animportant mechanism of POC. POC has been reported toinhibit the activation and postischemic dysfunction of thecoronary vascular endothelium. In addition, generation ofproinflammatory cytokines was reduced by POC [39].Preliminary studies by Granfeldt et al. [40] suggest thatPOC inhibits the inflammatory response to the reperfusionmediated by neutrophils. In support of this notion, POC hasbeen reported to inhibit the adherence of neutrophils tothe coronary vascular endothelium and the accumulation inthe extravascular and intravascular spaces. One of the keytriggers of POC, notably adenosine, has potent anti-inflammatory and antineutrophil effects. The results re-ported in the present paper support a similar anti-inflammatory mechanism for POC in the intestinal I/R. Inaddition, MDA data showing a reduction in membrane lipidperoxidation by POC may suggest another mechanism,namely the reduction of the oxidant generation, which hasalso been reported in the heart. However, whether the

126 I. Sengul et al.

source of oxidants was neutrophils or parenchymal tissue isnot known either for the heart or for the other tissues. Theplasma MDA levels in both POC modalities tended to belower than in control, but this was not a significant differ-ence. The mechanisms of POC may not be associated withthe oxygen radical reduction in this model. It may berelated to the reduction in inflammatory processes, incontrast to the heart. This is supported by the data.

In terms of adverse effects of POC, increased infarct sizehas been reported for very short durations of ischemia onlyand none in the models of more prolonged ischemia.Potential adverse event is damage to the endothelium atthe site of occlusionereocclusion. But no clinical adverseevents have been reported yet.

Our data showed that six cycles of POC were moreeffective than three cycles; however, the effects of varia-tion in the duration of these cycles were not investigated inthe present study. One mechanism of protection is relatedto decreasing the MDA levels of intestinal tissue, which is anindirect indicator of the lipid peroxidation, and thedecreasing the mucosal injury, inflammation, and totalhistopathological injury scores. However, no significantgroup difference was detected in regard to hemorrhage/hyperemia. This may be because the mucosa is moresusceptible to the duration of ischemia than the submu-cosal tissue. For example, the vascular endotheliumsustains injury prior to the underlying vascular musculartissue and myocardium. Temporal progression of vascularinjury for the heart may be different from that for theintestines, since the latter is not a highly metabolic tissue.

Therefore, we conclude that increasing the number ofcycles of POC from three to six increases its efficacy inreducing the mesenteric oxidant generation, lipid perox-idation, and neutrophil accumulation. Future studies arerequired that should include other algorithms (differentcycles) and longer durations of occlusion to answer thequestion about the threshold of the phenomenon.

Summary and Conclusion

POC is more clinically applicable than IPC when a widegroup of patients are thought as potentially treatable. Incontradistinction to IPC, POC can be applied during thereperfusion at the point of service for catheter-basedreperfusion [41], cardiac surgery [42,43], and organtransplantation, so it may have widespread clinicalapplications compared to the limited applications of IPC.The results of the present study may give rise to thepossible application of POC in fields such as revasculari-zation of the ischemic bowel primarily, bowel resectionand reanastomosis, intestinal organ transplantation, orother surgical procedures. Therefore, more advanced,sophisticated, and multicentered experimental and clin-ical studies are essential to optimize POC for applicationto the intestinal tract.

Acknowledgments

We would like to thank our colleagues, Oguzhan Ustaogluand Selim Temel, deeply and sincerely for their valuable

contributions to the manuscript. In addition, we want tothank sincerely Serkan Erkan, Mustafa Basar, Huseyin Ustun(the Chairman of Department of Pathology), and thepersonnel of the Pathology Laboratory, Animal Laboratory,and Laboratory of Biochemistry of Ankara Education andResearch Hospital for their assistance. This study was partlyfunded by Joint-Stock Company of Fako Medicines ActavisGroup, Island.

References

[1] Murry CE, Jennings RB, Reimer KA. Preconditioning withischemia: a delay of lethal cell injury in ischemic myocardium.Circulation 1986;74:1124e36.

[2] Zhao ZQ, Corvera JS, Halkos ME, Kerendi F, Wang NP,Guyton RA, et al. Inhibition of myocardial injury by ischemicpostconditioning during reperfusion: comparison withischemic preconditioning. Am J Physiol Heart Circ Physiol2003;285:H579e88.

[3] Sun K, Liu ZS, Sun Q. Role of mitochondria in cell apoptosisduring hepatic ischemiaereperfusion injury and protectiveeffect of ischemic postconditioning. World J Gastroenterol2004;10:1934e8.

[4] Xing B, Chen H, Zhang M, Zhao D, Jiang R, Liu X, et al.Ischemic postconditioning protects brain and reduces inflam-mation in a rat model of focal cerebral ischemia/reperfusion.J Neurochem 2008;105:1737e45.

[5] Eldaif SM, Deneve JA, Wang NP, Jiang R, Mosunjac M,Mutrie CJ, et al. Attenuation of renal ischemiaereperfusioninjury by postconditioning involves adenosine receptor andprotein kinase C activation. Transpl Int 2010;23:217e26.

[6] Park JW, Kang JW, Jeon WJ, Na HS. Postconditioning protectsskeletal muscle from ischemiaereperfusion injury. Microsur-gery 2010;30:223e9.

[7] Moon JG, Lim HC, Gye MR, Oh JS, Park JW. Postconditioningattenuates ischemiaereperfusion injury in rat skin flap.Microsurgery 2008;28:531e7.

[8] Ferencz A, Takacs I, Horvath S, Ferencz S, Javor S, Fekecs T,et al. Examination of protective effect of ischemic post-conditioning after small bowel autotransplantation. Trans-plant Proc 2010;42:2287e9.

[9] Santos CH, Gomes OM, Pontes JC, Miiji LN, Bispo MA. Theischemic preconditioning and postconditioning effect on theintestinal mucosa of rats undergoing mesenteric ischemia/r-eperfusion procedure. Acta Cir Bras 2008;23:22e8.

[10] Yang XM, Proctor JB, Cui L, Krieg T, Downey JM, Kohen M.Multiple, brief coronary occlusions during early reperfusionprotect rabbit hearts by targeting cell signaling pathways. JAm Coll Cardiol 2004;44:1103e10.

[11] Kin H, Zatta AJ, Lofye MT, Amerson BS, Halkos ME, Kerendi F,et al. Postconditioning reduces infarct size via adenosinereceptor activation by endogenous adenosine. Cardiovasc Res2005;67:124e33.

[12] Argaud L, Gateau-Roesch O, Raisky O, Loufouat J, Robert D,Ovize M. Postconditioning inhibits mitochondrial permeabilitytransition. Circulation 2005;111:194e7.

[13] Mykytenko J, Reeves JG, Kin H, Wang NP, Zatta AJ, Jiang R,et al. Persistent beneficial effect of postconditioning againstinfarct size: role of mitochondrial K(ATP) channels duringreperfusion. Basic Res Cardiol 2008;103:472e84.

[14] Serviddio G, Di Venosa N, Federici A, D’Agostino D, Rollo T,Prigigallo F, et al. Brief hypoxia before normoxic reperfusion(postconditioning) protects the heart against ische-miaereperfusion injury by preventing mitochondria peroxideproduction and glutathione depletion. FASEB J 2005;19:354e61.

Intestinal postconditioning 127

[15] Bopassa JC, Ferrera R, Gateau-Roesch O, Couture-Lepetit E,Ovize M. PI 3-kinase regulates the mitochondrial transitionpore in controlled reperfusion and postconditioning. Car-diovasc Res 2006;69:178e85.

[16] Yang XM, Philipp S, Downey JM, Cohen MV. Postconditioning’sprotection is not dependent on circulating blood factors orcells but involves adenosine receptors and requires PI3-kinaseand guanylyl cyclase activation. Basic Res Cardiol 2005;100:57e63.

[17] Sun HY, Wang NP, Kerendi F, Halkos ME, Kin H, Guyton RA,et al. Hypoxic postconditioning reduces cardiomyocyte loss byinhibiting ROS generation and intracellular Ca2þ overload. AmJ Physiol Heart Circ Physiol 2005;288:H1900e8.

[18] Ovize M, Baxter GF, Di Lisa F, Ferdinandy P, Garcia-Dorado D,Hausenloy DJ, et al. Postconditioning and protection fromreperfusion injury: where do we stand? Position paper fromthe Working Group of Cellular Biology of the Heart of theEuropean Society of Cardiology. Cardiovasc Res 2010;87:406e23.

[19] Zaugg M, Lucchinetti E, Spahn DR, Pasch T, Garcia C,Schaub MC. Differential effects of anesthetics on mitochon-drial K(ATP) channel activity and cardiomyocyte protection.Anesthesiology 2002;97:15e23.

[20] Morita Y, Murakami T, Iwase T, Nagai K, Nawada R, Kouchi I,et al. K(ATP) channels contribute to the cardioprotection ofpreconditioning independent of anaesthetics in rabbit hearts.J Mol Cell Cardiol 1997;29:1267e76.

[21] Hunter MI, Nlemadim BC, Davidson DL. Lipid peroxidationproducts and antioxidant proteins in plasma and cerebrospinalfluid from multiple sclerosis patients. Neurochem Res 1985;10:1645e52.

[22] Mihara M, Uchiyama M. Determination of malonaldehydeprecursor in tissues by thiobarbituric acid test. Anal Biochem1978;86:271e8.

[23] Chiu CJ, McArdle AH, Brown R, Scott HJ, Gurd FN. Intestinalmucosal lesion in low-flow states. I. A morphological, hemo-dynamic, and metabolic reappraisal. Arch Surg 1970;101:478e83.

[24] Moore FA. The role of the gastrointestinal tract in postinjurymultiple organ failure. Am J Surg 1999;178:449e53.

[25] Xu DZ, Lu Q, Kubicka R, Deitch EA. The effect of hypoxia/r-eoxygenation on the cellular function of intestinal epithelialcells. J Trauma 1999;46:280e5.

[26] Deem RL, Shanahan F, Targan SR. Triggered human mucosal Tcells release tumour necrosis factor-alpha and interferon-gamma which kill human colonic epithelial cells. Clin ExpImmunol 1991;83:79e84.

[27] McCallion K, Wattanasirichaigoon S, Gardiner KR, Fink MP.Ischemic preconditioning ameliorates ischemia- andreperfusion-induced intestinal epithelial hyperpermeability inrats. Shock 2000;14:429e34.

[28] Liu KX, Li YS, Huang WQ, Chen SQ, Wang ZX, Liu JX, et al.Immediate postconditioning during reperfusion attenuatesintestinal injury. Intensive Care Med 2009;35:933e42.

[29] Dos Santos CH, Pontes JC, Gomes OM, Miiji LN, Bispo MA.Evaluation of ischemic postconditioning effect on mesenteric

ischemia treatment: experimental study in rats. Rev Bras CirCardiovasc 2009;24:150e6.

[30] Li YS, Wang ZX, Li C, Xu M, Li Y, Huang WQ, et al. Proteomicsof ischemia/reperfusion injury in rat intestine with andwithout ischemic postconditioning. J Surg Res 2010;164:e173e80.

[31] Chu WW, Nie L, He XY, Yan AL, Zhou Y, Wu GL, et al. [Changeof cytochrome c in postconditioning attenuating ische-miaereperfusion-induced mucosal apoptosis in rat intestine].Sheng Li Xue Bao 2010;62:143e8.

[32] Rosero O, Onody P, Stangl R, Hegedus V, Lotz G, Blazovics A,et al. [Investigation of postconditioning in intestinal ische-miaereperfusion experimental models]. Magy Seb 2011;64:28e36.

[33] Ding JT, Zhang LY. Protective effects of ischemic post-conditioning on intestinal mucosa barrier function in rabbitswith crush injury of hind limb: an experimental study. Chin JTraumatol 2011;14:92e5.

[34] Bretz B, Blaze C, Parry N, Kudej RK. Ischemic postconditioningdoes not attenuate ischemiaereperfusion injury of rabbitsmall intestine. Vet Surg 2010;39:216e23.

[35] Halkos ME, Kerendi F, Corvera JS, Wang NP, Kin H, Payne CS,et al. Myocardial protection with postconditioning is notenhanced by ischemic preconditioning. Ann Thorac Surg 2004;78:961e9.

[36] Cohen MV, Yang XM, Downey JM. Acidosis, oxygen, and inter-ference with mitochondrial permeability transition poreformation in the early minutes of reperfusion are critical topostconditioning’s success. Basic ResCardiol 2008;103:464e71.

[37] Inserte J, Barba I, Hernando V, Garcia-Dorado D. Delayedrecovery of intracellular acidosis during reperfusion preventscalpain activation and determines protection in post-conditioned myocardium. Cardiovasc Res 2009;81:116e22.

[38] Kin H, Zhao ZQ, Sun HY, Wang N-P, Corvera JS, Halkos ME, et al.Postconditioning attenuates myocardial ischemiaereperfusioninjury by inhibiting events in the early minutes of reperfusion.Cardiovasc Res 2004;62:74e85.

[39] Kin H, Wang NP, Mykytenko J, Reeves J, Deneve J, Jiang R,et al. Inhibition of myocardial apoptosis by postconditioning isassociated with attenuation of oxidative stress-mediatednuclear factor-kappa B translocation and TNF alpha release.Shock 2008;29:761e8.

[40] Granfeldt A, Jiang R, Wang NP, Mykytenko J, Eldaif S,Deneve J, et al. Inhibition of neutrophils is critical to thein vivo cardioprotection of postconditioning. Circulation 2008;118:S403 (18_MeetingAbstracts).

[41] Yang XC, Liu Y, Wang LF, Cui L, Wang T, Ge YG, et al.Reduction in myocardial infarct size by postconditioning inpatients after percutaneous coronary intervention. J InvasiveCardiol 2007;19:424e30.

[42] Li B, Chen R, Huang R, Luo W. Clinical benefit of cardiacischemic postconditioning in corrections of tetralogy of Fallot.Interact Cardiovasc Thorac Surg 2009;8:17e21.

[43] Luo W, Li B, Chen R, Huang R, Lin G. Effect of ischemic post-conditioning in adult valve replacement. Eur J CardiothoracSurg 2008;33:203e8.