Cardiac Effect of Ischemic Preconditioning and Heparin FollowingIntestinal Ischemia and Reperfusion in Rats

R. Saurima, M.K. Koikeb, W.G.S. Bonservizia, G.A.A. Felixa, S.M. Silvaa, M.O. Tahac,and E.F.S. Monteroc,d,*aEscola Paulista de Medicina, Federal University of São Paulo; bLaboratory of Clinical Emergencies (LIM 51); cOperative Techniqueand Experimental Surgery Division, Escola Paulista de Medicina, Federal University of São Paulo; and dLaboratory of SurgicalPhysiopathology (LIM 62), Department of Surgery, University of São Paulo, Federal University of São Paulo, Brazil

0041-1345/1http://dx.doi

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ABSTRACT

To study the role of heparin and ischemic preconditioning (IPC) in cardiac injury afterintestinal ischemia (I) and reperfusion (R), 54 rats underwent 60 minutes of I, which wasproduced by occlusion of the superior mesenteric artery, and/or 120 minutes of R. The IPCgroup had the I procedure stimulation for 5 minutes and R for 10 minutes. The controlgroup was subjected to sham surgery only, and the other groups were injected with salinesolution (SS; 0.1 mL) or heparin (100 IU/kg) via the inferior cava vein 5 minutes before Iand 5 minutes before R and 55 minutes after the R begins in I-R groups. In all animals,cardiac samples were stained with hematoxylin and eosin for optical microscopy analysis,and other sample was processed for lipid peroxidation determination. In I-R groups, bothheparin and IPC showed significant protection compared to the SS group; conversely, inanimals subjected only to I, no protection was observed. Moreover, when heparin wasassociated with IPC, I-R protection was compromised and the ischemic injury increased.Data showed that IPC and heparin attenuated cardiac dysfunction caused by intestinal Iand I-R, but when used in association did not show beneficial effects.

*Address correspondence to Edna Frasson de Souza Montero,Al Espada, 134-RES, Onze, 06540-395, Alphaville, Santana deParnaíba, Brazil. E-mail: edna.montero@gmail.com

INTESTINAL ISCHEMIA (I) followed by reperfusion(R) leads to activation of circulating leukocytes that

trigger a local followed by a systemic microcirculatoryinflammatory response [1], oxygen free radical production[2], and multiple-organ failure as a final event. Therefore,intestinal I-R injury not only has local impact but alsocompromises distant organs. Horton and White [3]showed cardiac contractile depression as early as 2 hoursafter I-R in small intestine after superior mesenteric arteryocclusion.Ischemic preconditioning (IPC), which involves a brief

period of I and R preceding the sustained I of the organ [4],increases expression of intestinal aldose reductase toremove the reactive oxygen species, promotes down-regulation of nuclear factor-kB, reduces leukocyte-mediated tissue injury, and improves macrohemodynamicsand intestinal microcirculation, inducing systemic beneficialeffects [5]. The heart could be protected by subjecting aremote organ or tissue, such as kidney, small intestine, liver,or limb, to short episodes of I-R, a phenomenon known asremote IPC [6], and current evidence suggests that humoral

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mediators are released by the remote organ into circulationand exert their effects on the heart [7].Several therapeutic innovations for the prevention of

intestinal I-R-induced injury have been studied, showingvaried effects. Administration of heparin attenuated in-testinal dysfunction caused by I and I-R [8] and decreasedremote organ injury on the lung and heart after infrarenalaorta clamping in a rat model [9]. These effects are asso-ciated with the ability of heparin to down-regulate tumornecrosis factor-a (TNF-a)-induced leukocyte rolling,adhesion and migration into gut tissues [10], as well as theactivity and release of leukocyte enzymes [11] andapoptosis [12].The mechanism that both IPC and heparin protect

remote organs in I-R injury, and their associated effects inlocal and distant response to I-R injury are not totally clear.Therefore, this study was designed to evaluate the effect of

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Transplantation Proceedings, 46, 1852e1856 (2014)

CARDIAC EFFECT OF ISCHEMIC PRECONDITIONING 1853

these associated strategies on rat heart damage after intes-tinal I or I followed by R.

METHODS

The experimental protocol was submitted and approved by UNI-FESP Ethics Committee on Animal Research, protocol number0362/09. Adult, male Wistar rats (n ¼ 54) were used; they weighedbetween 250 and 350 g and were obtained from the Center forExperimental Models Development in Medicine and BiologyeFederal University of São Paulo. The animals were kept understandard conditions of light, humidity, temperature, and nutrition,and 12 hours before the experiment the supply of solid food wasrestricted.

After weighing, animals were anesthetized with a combination ofxylazine (10 mg/kg) and ketamine (60 mg/kg) intramuscularly.Midline laparotomy was performed, and the superior mesentericartery was dissected under surgical microscope (10-fold increase)using microsurgical instruments.

The animals were divided randomly into 9 groups with 6 rats ineach group: control group, sham surgery; I þ SS group, animalstreated with saline solution þ intestinal I; I-R þ SS group, animalstreated saline solution þ intestinal I and R; I þ heparin group,animals treated with heparin þ intestinal I; I-R þ heparin group,animals treated with heparin þ intestinal I and R; I þ SS þ IPCgroup, animals treated with saline solution þ intestinal IPC þ in-testinal I; I-R þ SS þ IPC group, animals treated with salinesolution þ intestinal IPC þ intestinal I and R; I þ heparin þ IPCgroup, animals treated with heparin þ intestinal IPC þ intestinal I;I-R þ heparin þ IPC group, animals treated with heparin þ in-testinal IPC þ intestinal I and R.

Control group animals underwent only laparotomy without I andR. In the other groups, the superior mesenteric artery was isolatedand repaired with 4-0 cotton thread. Vascular occlusion was ob-tained by applying microsurgical vascular clamp.

I and I-R Protocol

Rats were submitted to 60 minutes of I (groups I þ SS; I þ heparin;I þ SS þ IPC; I þ heparin þ IPC) followed by 120 minutes of R(groups I-R þ SS; I-R þ heparin; I-R þ SS þ IPC; I-R þ heparin þIPC).

IPC Protocol

Groups treated with IPC (I þ SS þ IPC; I þ heparin þ IPC; I-R þSS þ IPC; I-R þ heparin þ IPC) were subjected to 5 minutes of Iand 10 minutes of R before the prolonged I, by clamping the su-perior mesenteric artery.

Heparin Protocol

Heparin (100 IU/kg) or saline solution at the similar volume(0.1 mL) was administered 5 minutes before I and 5 minutes beforeR and after 55 minutes of R in I and R groups. Heparin has beenstudied in our laboratory looking at other organs with this samedose [8,9,12]. The dose was chosen based on a previously workpublished by Hirsh et al [13]. They report the half-life of heparinincreases from approximately 60 minutes with a bolus of 100 U/kg,justifying our protocol with 3 doses, during the experiment.

Following the procedures, animals were euthanized underanesthesia to surgically remove heart tissue samples for histologicanalysis and quantification of thiobarbituric acid reactive substances(TBARS).

Histologic Analysis

Samples of cardiac tissue excised for morphologic examination werefixed in 10% formalin and embedded in paraffin and sections of 3 mmwere done. The slides were stained with hematoxylin and eosin andqualitatively evaluated under an opticalmicroscope (ZeissAxio ImageA2, Oberkochen, Germany) by an experienced pathologist kept blindto the different groups. At least 10 fields of each slide were randomlychosen and analyzed. The cardiac histologic score was based on thescale from 0 to 5, as follows: 0 for injury missing, 1 for light injury(contraction bands and scattered pyknosis), 2 for mild injury(contraction bands, and pyknosis karyorrhexis), 3 for moderate injury(contraction band, pyknosis, and hypereosinophilia karyorrhexis), 4for moderate to intense injury (contraction band, pyknosis, hyper-eosinophilia, karyorrhexis and vacuolization), and 5 for intense injury(all others, and presence of edema or inflammation) [14].

TBARS Analysis

Cardiac tissue sample was frozen at �80�C. After the tissue sampleswere homogenized in 1 mL of 1.15% KCl with sonicator (PolytronPT3100) and used for determination of malondialdehyde (MDA).The cell membrane lipid peroxidation was determined by themethod of TBARS, which measures the amount of MDA derivativeof lipid peroxidation, and the value was expressed in nanomoles permilligram of protein (nmol/mg protein). For this purpose, afterhomogenization, aliquots were centrifuged at 10,000 revolutions perminute for 20 minutes at 4�C (Eppendorf Centrifuge 5804,Hamburg, Germany). Sodium dodecyl sulfate (100 mL) was addedto 8.1% 750 mL of 20% acetic acid and 750 mL of thiobarbituric acidto 8%. The mixture was heated for 50 minutes at 95�C. After the setperiod, samples were analyzed 200 mL of 532-mn spectrophotom-eter (Multiscan Ex, MTX Labsystems, Va, United States). All an-alyses were performed in duplicate.

Statistical Analysis

Data are presented as mean � standard deviation. Analysis ofvariance test was used for comparison among groups. Statisticalsignificance were considered for P � .05.

RESULTS

Morphologic analysis showed that cardiac tissue sufferedinjury because of intestinal I (2.0 � 1.3) and I-R (3.5 � 0.5)compared to control group (grade 0; P < .001). It was alsoobserved after R that heparin (1.3 � 0.5) or IPC (1.7 � 0.7)modulates this lesion (P < .001; P < .007, respectively;Fig 1). However, the association of both strategies showeddeleterious effect, as it can be observed in Table 1. Simi-larly, lipidic peroxidation was reduced by IPC or heparin,but not when these strategies were associated (Table 1).

DISCUSSION

These results suggest that IPC and heparin attenuatedcardiac dysfunction caused by intestinal I and I-R, but whenused in association did not show beneficial effects.Intestinal I-R is a life-threatening clinical complication that

can lead to systemic inflammatory response syndrome for thetranslocation of inflammatory mediators, bacteria, andendotoxin from injured intestines and by immune systemactivation [15], but the exact pathophysiologic mediators

Table 1. Values of MDA and Histologic Scores of the CardiacTissue (Expressed as Mean ± Standard Deviation)

Groups MDA (ng/mg of Protein) Histology (Score 0 to 5)

Control 1.54 � 0.2 0I þ saline solution 2.05 � 0.9 2.0 � 1.3I-R þ saline solution 2.15 � 0.2 3.5 � 0.5I þ IPC þ saline solution 1.95 � 0.3 2.1 � 0.9I-R þ IPC þ saline solution 1.60 � 0.3 1.7 � 0.7I þ heparin 2.16 � 0.4 2.8 � 1.2I-R þ heparin 1.60 � 0.3 1.3 � 0.5I þ IPC þ heparin 2.11 � 0.2 3.7 � 0.5I-R þ IPC þ heparin 2.15 � 0.2 3.5 � 0.5

Abbreviations: MDA, malondialdehyde; I, ischemia; R, reperfusion; IPC,ischemic preconditioning.

1854 SAURIM, KOIKE, BONSERVIZI ET AL

involved are still unclear. Horton and White demonstratedthat one of the mechanisms involved in cardiac damage is theproduction of reactive oxygen species and lipid peroxidationin cardiac cell membranes [16], contributing to endothelialdysfunction and leading to reduced nitric oxide, increasedproinflammatory cytokine production, and adhesion mole-cule expression [17].Studying intestinal I-R, Taha and coworkers observed

that both heparin [8] and IPC [18] attenuated gut dysfunc-tion caused by I-R, preserving jejunal enteric nerves andorgan motility [8,18], but when used together did notdemonstrate additional protection. Based on an experi-mental model with a similar design, our study showedreduction in the systemic damage of this process bydecreasing the structural lesion and lipid peroxidation in theheart of rats subjected to intestinal I-R.IPC comprises two phases of protection: the first phase,

termed “early,” protects tissue for an hour or 2 and thenwanes; the second phase, “delayed,” appears 24 hours afterthe IPC protocol and can last for 3 days [19]. The protectiveeffects of IPC on the small intestine is correlated with in-hibition of epithelial cell apoptosis [20], inhibition of P-selectin expressionda critical step for leukocyte rolling andneutrophil sequestration [21] and in the first phase severalmediators play a crucial role in the protective phenomenon,including adenosine [22], nitric oxide [23], and protein ki-nase C [24].The reduction of intestinal damage by the effect of these

protective mechanisms could explain the decrease in cardiaclesion in animals pretreated with IPC by decreasing

Fig 1. Histology from (A) I-R þ saline so-lution group (rat 5); (B) I-R þ heparin group(rat 4); (C) I-R þ IPC þ saline solutiongroup (rat 3); and (D) I-R þ IPC þ heparingroup (rat 3). I, ischemia; R, reperfusion,IPC, ischemic preconditioning.

translocation of noxious stimuli. However, beyond the in-testinal effect, occlusion of the superior mesenteric arteryfor a short period prior to sustained I of the gut causes aheart-protective effect, which is called remote IPC phe-nomenon [6]. Release of biochemical messengers into cir-culation and activation of nerve pathways are described aspossible mediators of protection, and Kang et al additionallyrevealed that the cardioprotective effect of IPC could beattributed to endogenous nitric oxide production [25].Heparin is known to have a variety of activities, not only

effects on blood coagulation. Anti-inflammatory applica-tions of heparin include the inhibition of elastase releasefrom neutrophils and the adhesion of these cells to vascularendothelium [11]. Studies have suggested that heparin hasan antioxidant effect and inhibits apoptosis in several cell

CARDIAC EFFECT OF ISCHEMIC PRECONDITIONING 1855

types, protecting them from I-R lesions [26]. Heparin andderived compounds are related to the binding of growthfactors, especially FGF-1 and FGF-2, to its receptors or tothe activation of signal transduction [27], and can alsoenable the reduction of the enzymatic degradation of thesesubstances [28]. Administration of heparin-bindingepidermal growth factor-like growth factor (HB-EGF) de-creases neutrophileendothelial cell interactions [29], reac-tive oxygen species production [30], and levels of theproinflammatory cytokines TNF-a, interleukin-6, andinterleukin 1-b after intestinal I-R injury [31].IPC and heparin used together demonstrated protection

in the intestine subjected to I-R, but did not offer additionalgain over the individual use of each technique. Therefore,improvement was expected in systemic lesion by decreasedlocal production and, consequently, translocation of injurymediators, increased lipid peroxidation, and histologic car-diac damage were observed.Warzecha et al, in an I-R-induced acute pancreatitis

model, showed an increase in pancreatic damage andplasma level of lipase, amylase, and interleukin-1b andinterleukin-10 in groups treated with heparin and IP in as-sociation when compared with heparin or IPC solely [32].This study showed that the application of heparin in thegroups submitted to the IPC decreased the levels ofd-dimer, a marker of fibrinolysis, indicating inhibition ofearly activation of fibrinolysis due to IPC, and Williams-Pritchard et al demonstrated that ligand inhibitors of HB-EGF eliminate the cardioprotective effects of IPC [33].The possible mechanism involved in competitive interactionon pathways shared by heparin and IPC could probablyexplain the negative effects of this association in heart ofrats subjected to intestinal I and I-R.

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