Experimental acute myocardial infarction in rats: HIF-1α, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin

G

A

Eee

ASa

b

c

d

e

f

g

a

ARRAA

KEECHMR

I

nrlE2P

0h

ARTICLE IN PRESS Model

CTHIS-50681; No. of Pages 11

Acta Histochemica xxx (2013) xxx– xxx

Contents lists available at SciVerse ScienceDirect

Acta Histochemica

jou rna l h o mepage: www.elsev ier .de /ac th is

xperimental acute myocardial infarction in rats: HIF-1�, caspase-3,rythropoietin and erythropoietin receptor expression and the cardioprotectiveffects of two different erythropoietin doses

ysel Guven Baglaa,∗ , Ertugrul Ercanb, Halil Fatih Asgunc, Meltem Ickina, Feriha Ercand, Ozlem Yavuze,uat Bagla f, Askin Kaplang

Department of Histology and Embryology, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, TurkeyDepartment of Cardiology, Faculty of Medicine, Medicalpark Hospital, Izmir University, Izmir, TurkeyDepartment of Cardiovascular Surgery, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, TurkeyDepartment of Histology and Embryology, Faculty of Medicine, Marmara University, Istanbul, TurkeyDepartment of Medical Biochemistry, Balikesir University, Faculty of Medicine, Balikesir, TurkeyCanakkale Provincial Directorate of Health, Canakkale, TurkeyFamily Medicine, Istanbul Aile Hekimligi, Istanbul, Turkey

r t i c l e i n f o

rticle history:eceived 28 November 2012eceived in revised form 19 January 2013ccepted 23 January 2013vailable online xxx

eywords:rythropoietinrythropoietin receptoraspase 3ypoxia inducible factor 1�yocardial infarction

at

a b s t r a c t

The cardioprotective effects of two different doses of erythropoietin administration were analyzedin rats with experimental myocardial infarction. None, saline, standard-dose (5000 U kg−1) and high-dose (10,000 U kg−1) of human recombinant erythropoietin alpha were administered intraperitoneallyin Wistar rats with myocardial infarction induced by coronary artery ligation. Infarct sizes measuredafter triphenyltetrazolium chloride staining, levels of biochemical markers, histopathology examined bylight and electron microscopy, and immunohistochemical expressions of erythropoietin, erythropoie-tin receptor, hypoxia inducible factor-1� and caspase-3, were analyzed. Lower scores of infarction andhemorrhage, lower number of macrophages and higher score of vascularization surrounding the infarctarea were observed in the erythropoietin administered groups (p < 0.05). Erythropoietin administrationafter myocardial infarction reduced the area of infarction and hemorrhage. There were hypoxia induciblefactor-1� and caspase-3 expressions in the marginal area, and erythropoietin and erythropoietin receptorexpression in both marginal and normal areas (p < 0.001). Vascularization, erythropoietin expression inthe normal area and vascular erythropoietin expression were positively correlated with human erythro-
poietin levels. The cardioprotective effects of erythropoietin treatment were independent of endogenouserythropoietin/erythropoietin receptor activity. Moreover exogenous erythropoietin treatment did notsuppress endogenous erythropoietin. Erythropoietin administration after myocardial infarction reducedcaspase 3 expression (apoptotic activity) and induced neovascularization around the infarct area. Highererythropoietin administration did not provide an additional benefit over the standard-dose in myocardialprotection.
ntroduction

Erythropoietin (Epo) is a glycoprotein hormone essential forormal erythrocyte production in bone marrow. It is released fromenal peritubular cells and various extrarenal tissues including:iver, spleen, brain, lungs, bone marrow, and reproductive organs.

Please cite this article in press as: Guven Bagla A, et al. Experimental atin and erythropoietin receptor expression and the cardioprotective effechttp://dx.doi.org/10.1016/j.acthis.2013.01.005

po induces erythropoiesis under hypoxic conditions (Sasaki et al.,000; Chong et al., 2002; Jelkmann, 2004; Marzo et al., 2008;aschos et al., 2008). Interaction of Epo with its receptor decreases

∗ Corresponding author.E-mail address: [email protected] (A. Guven Bagla).

065-1281/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.acthis.2013.01.005

© 2013 Elsevier GmbH. All rights reserved.

programmed death of erythroid progenitor cells and promotes theirdifferentiation in bone marrow (Fisher et al., 1996; Sasaki et al.,2000).

The protective effects of Epo against tissue ischemia are medi-ated by erythropoietin receptor (EpoR) (Brines et al., 2004). Inaddition to erythroid progenitor cells, a varied group of cellsincluding neurons, endothelial cells, vascular smooth muscle cells,and cardiac myocytes, express EpoR (Anagnostou et al., 1994;Digicaylioglu et al., 1995; Akimoto et al., 2000; Ogilvie et al.,

cute myocardial infarction in rats: HIF-1�, caspase-3, erythropoie-ts of two different erythropoietin doses. Acta Histochemica (2013),

2000; Ammarguellat et al., 2001; Digicaylioglu and Lipton, 2001;Tramontano et al., 2003; Jelkmann and Wagner, 2004; Marti,2004). The cardioprotective effects of Epo have become a topi-cal issue after detection of EpoR expression on cardiomyocytes

dx.doi.org/10.1016/j.acthis.2013.01.005


http://www.sciencedirect.com/science/journal/00651281

http://www.elsevier.de/acthis

mailto:[email protected]


ING Model

A

2 Histoc

(c2ee2trpatiaiape

hie2Estcr

M

mcmpsA

gctai(pi((fia

M

15desCl(tho

ARTICLECTHIS-50681; No. of Pages 11

A. Guven Bagla et al. / Acta

Marzo et al., 2008). The interaction of Epo with EpoR on theseells exerts protective effects against tissue ischemia (Junk et al.,002; Cai et al., 2003; Calvillo et al., 2003; Moon et al., 2003; Parsat al., 2003; Cai and Semenza, 2004; Lipsic et al., 2004; Sharplest al., 2004; Solaroglu et al., 2004; Wu et al., 2006; Guneli et al.,007). Epo inhibits apoptosis and limits infarct size as seen usingriphenyltetrazolium chloride (TTC) staining during ischemia andeperfusion through activation of various intracellular signalingathways, especially the PI3K-Akt pathway (Parsa et al., 2003). Epolso has anti-inflammatory, anti-oxidative, and angiogenic poten-ial (Paschos et al., 2008). The cardioprotective effects of Epo arendependent of its hematopoietic effects (Parsa et al., 2003). Similarntiapoptotic and cardioprotective effects of Epo, independent ofts hematopoietic effects, have been shown with carbamylated Epo,

non-erythropoietic derivative of Epo, and with helix B-surfaceeptide, a peptide mimicking the 3D structure of Epo (Fiordalisot al., 2005; Ueba et al., 2010; Ahmet et al., 2011).

Several studies regarding the cardioprotective effects of Epoave been tested using a similar standard dose of Epo treatment

ncluding 3000 U kg−1 or 5000 U kg−1 (Calvillo et al., 2003; Moont al., 2003; Parsa et al., 2003; Tramontano et al., 2003; Rui et al.,005). It remains to be clarified whether a higher-dose of thepo treatment may improve the cardioprotective effects over thetandard-dose treatment. In this study, cardioprotective effects ofwo different doses of Epo treatment were analyzed using bio-hemical, morphological, and immunohistochemical methods inats with myocardial infarction induced by coronary artery ligation.

aterials and methods

The study was approved by Gazi University Animal Experi-ents Local Ethics Committee (project number G.Ü.ET-08.059) and

onformed to the Directive 2010/63/EU of the European Parlia-ent on the protection of animals used for scientific purposes. All

rocedures on laboratory animals were performed in Gazi Univer-ity Laboratory Animals Care and Experimental Research Center,nkara, Turkey.

Male Wistar rats weighing 250–300 g were divided into fiveroups: Group 1: Rats without treatment and sacrificed 1 h afteroronary ligation (n = 8); Group 2: Rats receiving a single intraperi-oneal (i.p) injection of saline immediately after coronary ligationnd sacrificed 6 h after surgery (n = 7); Group 3: Rats receiv-ng a single i.p. injection of standard-dose (5000 U kg−1) Epohuman recombinant erythropoietin alpha) (Eprex 4000 IU/0.4 mLre-filled syringe, Janssen-Cilag AG, Schaffhausen, Switzerland)

mmediately after coronary ligation and sacrificed 6 h after surgeryn = 9); Group 4: Rats receiving a single i.p. injection of high-dose10,000 U kg−1) Epo immediately after coronary ligation and sacri-ced 6 h after surgery (n = 9); Group 5: Sham-operated control ratsnd sacrificed 6 h after surgery (n = 3).

yocardial ischemia model

Rats were anesthetized with 45 mg kg−1 ketamine (Alfamine0%, Alfasan International BV, Woerden, The Netherlands) and

mg kg−1 xylazine (Alfazyne 2%, Alfasan International BV, Woer-en, The Netherlands) administered intraperitoneally. Basallectrocardiograms of all rats were taken using a data acquisitionystem (MP 150 Data Acquisition System, BIOPAC Systems, Goleta,A, USA). Rats were intubated through tracheotomy and venti-

ated with room air using a volume controlled rodent ventilator


Inspira ASV, Harvard Apparatus, Holliston, MA, USA). Thoracotomyhrough the fourth intercostal space was performed to expose theeart. Left anterior descending coronary artery (LAD) ligation wasbtained by placing a 7–0 polypropylene suture around the space

PRESShemica xxx (2013) xxx– xxx

between the pulmonary artery and the left auricle. Cessation ofnormal contractions of the myocardium in the LAD perfusion areawas accepted as a marker of adequate LAD ligation. Following LADligation, saline, standard-dose erythropoietin and high-dose eryth-ropoietin were administered intraperitoneally in Groups 2, 3, and 4,respectively. The thoracotomy was closed without residual pneu-mothorax, and the tracheotomy was repaired after weaning fromthe ventilator. Control electrocardiograms of all rats were taken.ECG recordings taken after left anterior descending coronary artery(LAD) ligation showed ST segment changes indicating myocardialinfarction. Rats were allowed to recover in a warm and oxygen richcompartment until they were fully active, and then they were trans-ferred to their cages. No additional analgesia was required over theinitial anesthesia. None of the rats died before euthanasia.

Rats were fully anesthetized again with the same dose ofketamine and xylazine before euthanasia, and were re-intubatedvia the previous tracheotomy. The heart was exposed througha midline sternotomy and excised quickly after a blood samplewas withdrawn from the right atrium. The occurrence of acutemyocardial infarction in each rat was verified qualitatively usinga commercially available troponin T test kit (Trop T, Roche Diag-nostics, West Sussex, UK).

Biochemistry

Rat Epo, rat high sensitivity C-reactive protein (hsCRP), andrat cardiac troponin T (cTnT) levels in rat serum were measuredusing commercially available enzyme-linked immunosorbentassay (ELISA) rat sensitive kits (Cusabio Biotech, Newark, DE, USA,Catalog no: CSB-E07323r, CSB-E08618r, and CSB-E11305r, respec-tively) on a Model 680 microplate reader (Bio-Rad Laboratories,Hercules, CA, USA). The detection limits of Epo, hsCRP, and cTnTin rat serum were 0.2 mIU mL−1, 0.16 ng mL−1, and 15.6 pg mL−1,respectively. Human Epo levels in rat serum were measured usinga DRG EPO ELISA kit (DRG International, East Mountainside, NJ, USA,Catalog no: EIA-3646).

Light microscopy

The hearts were fixed in 10% buffered formalin, and processedfor embedding in paraffin wax by routine protocols and cut into5-�m-thick sections by a microtome. The sections were stainedusing hematoxylin and eosin (H&E) and examined using a photomi-croscope (Axioskop 40 Microscope and AxioCam ICc3 MicroscopeCamera, Carl Zeiss, Göttingen, Germany). Five sections were ran-domly chosen from the mid-ventricular level of the heart in eachanimal. From each section, five areas were randomly selected forthe histopathological examination. The presence of vascularizationaround the infarct area, the score of infarction area, hemor-rhage, inflammatory cell infiltration (increase in neutrophils), andthe number of macrophages were assessed. Macrophages werecounted (40× objective) in five different areas surrounding theinfarct area.

Semi-quantitative analysis

Semi-quantitative analysis of the infarct size in the left ventri-cle, hemorrhage, and leukocyte increase were scored as: none (0),weak (1), moderate (2), strong (3) and very strong (4). The semi-quantitative lesion scoring system was adapted from Azevedo Filhoet al. (2004). Each section received a score according to the per-centage of the infarct size in the left ventricle (score 0, 1, 2, 3 and


4). Score 0 corresponded to the absence of infarct; score 1 (weak)corresponded to infarct size of 1–25% of the area of the segment;score 2 (moderate) corresponded to infarct size of 26–50%; score 3(strong) corresponded to infarct size of 51–75%; and score 4 (very


ING Model

A

Histoc

st

ain((

T

dewTiSwEos

E

mmmfistatWsa(

I

v1plusfahecpHabpUpHSaPww



trong) corresponded to infarct size greater than 76% of the area ofhe segment.

Leukocyte increase was scored as none (0), weak (1), moder-te (2), strong (3) as follows: leukocyte neutrophils were countedn 10 random section areas surrounding the infarct area and theumber of cells were revealed as follows: none (0): absent; weak1): occasional (1–10 cells), moderate (2): focal (10–50 cells), strong3): focal (>50 cells) and/or diffuse.

TC staining

The hearts were stained with TTC for the delineation of myocar-ial infarction using the method described previously (Vivaldit al., 1985). The transverse heart slices at mid-ventricular levelere obtained freehand, and incubated in a 1% solution of

TC in phosphate buffer for 5 min at 37 ◦C, pH 7.4. The TTC-ncubated slices were photographed in a macroscope (Leica M125tereoscope, Leica Microsystems, Wetzlar, Germany). Photographsere processed using graphics editing software (Photoshop CS4

xtended, Adobe Systems, San Jose, CA, USA) to calculate the sizef infarct area, which was expressed as a fraction of the total cross-ectional area of the left ventricle.

lectron microscopy

The ultrastructure of heart tissue was analyzed using trans-ission electron microscopy from samples obtained from theid-ventricular level of the heart. Blocks of heart tissue speci-en approximately 1 mm3 were removed from each animal and

xed in 2.5% glutaraldehyde (pH 7.3) in 0.1 M phosphate-bufferedaline at 4 ◦C for 4 h. The specimens were postfixed in 1% osmiumetroxide (0.1 M), and dehydrated in a graded series of ethanol,nd embedded in epoxy resin. The epoxy resin blocks were sec-ioned using an Ultracut R microtome (Leica Microsystems GmbH,

etzlar, Germany). Ultrathin sections (70 nm thick) were contrasttained with uranyl acetate and lead citrate prior to examinationnd image recording under a transmission electron microscopeJEOL, 1200 EX II, TEM, Tokyo, Japan).

mmunohistochemistry

All sections were processed simultaneously to avoid day-to-dayariation of labeling efficiency. The tissue samples were fixed in0% formalin, and embedded in paraffin wax blocks using routinerotocols. Four-�m-thick sections were cut and mounted on poly--lysine-coated microscope slides. Sections were immunolabeledsing an indirect immunohistochemical horseradish peroxidasetaining procedure for the presence of Epo, EpoR, hypoxia inducibleactor (HIF)-1�, and caspase 3. The sections were deparaffinizednd rehydrated by passing through xylene, graded ethyl alco-ol (100%, 96%, and 70%), and bidistilled sterile water for 15 minach. Heat-induced antigens were retrieved using 0.01 mol L−1

itrate buffer (pH 6.0). The slides were quenched and washed inhosphate-buffered saline (PBS). Sections were treated with 3%2O2 and methanol for 30 min to block endogenous peroxidasectivity. Sections were washed in PBS, pH 7.4. Slides were incu-ated overnight at 4 ◦C with Epo (H-162) antibody (sc-7956, Rabbitolyclonal antibody, Santa Cruz Biotechnology, Santa Cruz, CA,SA) at 1:100 dilution, EpoR (H-194) antibody (sc5624, Rabbitolyclonal antibody, Santa Cruz Biotechnology) at 1:200 dilution,IF-1� (H1�67) antibody (sc-53546 Mouse monoclonal antibody,anta Cruz Biotechnology) at 1:100 dilution, and caspase 3 (CPP32)


ntibody (Rabbit polyclonal antibody, Diagnostic BioSystems,leasanton, CA, USA) at 1:500 dilution. After thorough washingith PBS, the sections were flooded with 5% H2O2 solution, rinsedith PBS (2× 5 min) and incubated with biotinylated polyvalent

PRESShemica xxx (2013) xxx– xxx 3

IgG (ready-to-use, Invitrogen, Life Technologies Corporation, Carls-bad, CA, USA) for 15 min. Sections were rinsed with PBS (2× 5 min)and incubated with a streptavidin-horseradish peroxidase complex(ready-to-use, Invitrogen, Life Technologies Corporation, Carlsbad,CA, USA) for 15 min. After rinsing with PBS (2× 5 min) again, sec-tions were incubated with chromogen substrate solution for 15 minfreshly prepared by dissolving 1 mg 3,3′-diaminobenzidine (DAB)(Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA) in1 mL of 0.05 M Tris–HCl buffer, pH 7.4, containing 1 mL of H2O2.After rinsing in distilled water, the sections were counterstainedwith Harris’s hematoxylin. For dehydration, each slide was soakedin graded alcohols (75%–80%–96%–2× 100%), cleared twice withxylene, and coverslipped with mounting medium. All antibodieswere rat specific. Negative controls for all groups were performedby replacing the primary antisera with PBS.

Semi-quantitative analysis of immunohistochemicalstaining evaluation

The immunostaining patterns were diffuse through the wholeseries of sections for caspase-3, HIF-1�, Epo and EpoR in normal andmarginal areas. The marginal area refers to the area surroundingthe infarction. None (0) = no detectable stain; (1) = weak to moder-ate staining; (2) = moderate to strong staining; (3) = strong to verystrong staining; (4) = very strong staining on the infarct area, themarginal area and the normal area separately.

The analyses of specimens in light microscopy, electronmicroscopy, and immunohistochemistry were performed by aresearcher blinded to the sample grouping.

Statistical analysis

Statistical analyses were performed using IBM SPSS Statisticsversion 19 (IBM Corporation, NY, USA). Results of rat weight,the infarct size in TTC staining, biochemical markers, and thenumber of macrophages were expressed as mean ± SD. Results oflight microscopy and immunohistochemistry were represented asstacked bar graphs. Kruskal–Wallis test was performed to ana-lyze statistical significance. Multiple comparisons between groupswere done using the Mann–Whitney U test with Bonferroni adjust-ment. Results of Group 5 were excluded from the tests done forinfarct size in TTC staining, the score of infarction, hemorrhage,leukocyte increase, number of macrophages, vascularization, andHIF-1�, Epo, EpoR and caspase-3 expression in the infarct area andthe marginal area, because the rats in this group had no infarc-tion induced by the coronary ligation. Spearman’s and Kendall’sRank Correlation tests were performed to measure the correla-tion between variables. p < 0.05 was considered to be a statisticallysignificant difference.

Results

Biochemistry

Results of rat weights, infarct sizes in TTC staining, and levels ofhuman Epo, rat Epo, cTnT, and hsCRP in rat serum are represented inTable 1. TTC staining is represented in Fig. 1. The greatest infarct sizeas seen in TTC staining was observed in Group 2 (37.11 ± 22.28%).Although the infarct sizes in TTC staining in the Epo administeredgroups were lower than Group 2, the difference was insignifi-


cant (p = 0.093). Human Epo level was significantly high in the Epoadministered groups (p < 0.001), and Group 3 and Group 4 werestatistically identical (p = 1). The levels of rat Epo, cTnT, and hsCRPwere similar in all groups.


ARTICLE IN PRESSG Model

ACTHIS-50681; No. of Pages 11

4 A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx– xxx

Table 1Infarct sizes in TTC staining and results of biochemical markers.

Group 1 (n = 8) Group 2 (n = 7) Group 3 (n = 9) Group 4 (n = 9) Group 5 (n = 3) p

Infarct size (%) 9.49 ± 9.34 37.11 ± 22.28 21.49 ± 10.58 25.55 ± 13.65 – 0.121Human Epo level (mIU/mL) 12.2 ± 3.31 12.49 ± 2.78 1398.1 ± 123.29 1379.03 ± 201.29 64.52 ± 88.18 <0.001*

Rat Epo level (mIU/mL) 23.47 ± 15.6 40.05 ± 75.18 7.71 ± 5.34 42.54 ± 41.66 14.21 ± 6.81 0.507cTnT level (pg/mL) 436.18 ± 340.3 319.23 ± 429.04 205.59 ± 132.89 583.8 ± 261.64 282.95 ± 146.05 0.226hsCRP level (ng/mL) 0.079 ± 0.047 0.067 ± 0.03 0.05 ± 0.026 0.06 ± 0.024 0.04 ± 0.02 0.371

c otein.

L

eancGw(a(mr(wtv(o

Ft

TnT: Cardiac troponin T, Epo: Erythropoietin, hsCRP: High sensitivity C-reactive pr* Human Epo level was significantly high in the Epo administered groups.

ight microscopy

The cardiomyocytes in the infarct areas revealed denseosinophilic cytoplasm and small condensed nuclei (Fig. 2And B). The scores of infarction size, hemorrhage, and theumber of macrophages in light microscopy were statisti-ally different between the groups, and were the highest inroup 2. The highest changes in histopathological examinationere observed in the left ventricular infarct area in Group 2

p < 0.001) (Figs. 2A and 3A). Epo administered groups showed decrease in the area of infarct sizes (Groups 3 and 4)p < 0.001) (Figs. 2A, B and 3A). Marked hemorrhage, widespread

yofilament disarray and loss were observed between infarctegions in some areas in Group 1 and Group 2 (p = 0.002)Figs. 2B–D and 3B). Increase in the number of neutrophilsas observed in Group 1 and Group 2, but it was statis-


ically insignificant (p = 0.197) (Figs. 2C, D and 4B). Markedascularization was seen around the infarct area in Group 3p = 0.014) (Figs. 2C and 3C). Statistical difference in the numberf macrophages was achieved between Group 2 and Group 4

ig. 1. TTC staining showing infarct areas. Group 1: 1 h after coronary artery ligation; Grohe time of ligation + 6 h after coronary artery ligation; Group 4: EPO 10,000 U kg−1 admin

(p = 0.027) (Fig. 4A). Morphological features appeared normal inGroup 5 (control group) (Fig. 2A–D).

In terms of the infarct size, hemorrhage, and the number ofneutrophils, no significant difference was observed between Epoadministered groups (Groups 3 and 4) (Figs. 3A, B and 4B).

Whereas vascularization, Epo expression in the normal area, andvascular Epo expression were positively correlated with humanEpo levels in rat serum (p = 0.006), the number of macrophages, thescore of infarct area, hemorrhage, and caspase-3 expression in themarginal area were negatively correlated with human Epo levels(p = 0.029, p = 0.013, p = 0.033, p = 0.025, respectively).

Electron microscopy

Electron microscopy showed a normal cell morphology andarrangement in Group 5 (Fig. 5A). Mild mitochondrial degeneration,


intracytoplasmic vacuolization, focal myofilament disarray, andfairly regular intercalated disks were observed in Group 1 (Fig. 5B).Severe mitochondrial degeneration, intracytoplasmic vacuoliza-tion, widespread myofilament disarray, and dilated intercalated

up 2: 6 h after coronary artery ligation; Group 3: EPO 5000 U kg−1 administered atistered at the time of ligation + 6 h after coronary artery ligation.




A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx– xxx 5

Fig. 2. The images represent the myocardium in light microscopy 1 h and 6 h after coronary artery ligation group: to which EPO was not injected; A, The infarct regions wereseen as marked areas (↔). B, In the infarct areas (I), the cardiomyocyte sarcoplasm was densely eosinophilic and nuclei showed decreased size and condensation. N: Normalarea. C, D, Marked hemorrhage (→), widespread myofilament disarray and loss were observed between infarct regions in some areas. Increase in the number of neutrophils(*) was observed. 6 h after coronary artery ligation and EPO 5000 IU/kg (6 Hour + EPO 5,000) and EPO 10,000 IU/kg (6 Hour + EPO 10,000) treated group; A–B: reductiono inophi5 rpholD

dmr

I

m

ae

1rnHai

tEiwmEG

f infarct areas (↔) were observed. The sarcoplasm of cardiomyocytes stains eos,000, hematoxylin–eosin staining (H&E). In the control group (A–B–C–D) these mo

= 50 �m.

isks were found in Group 2 (Fig. 5C). A few vacuoles, milditochondrial degeneration, focal myofilament disarray, and quite

egular intercalated disks were seen in Group 3 (Fig. 5D).

mmunohistochemistry

Expressions of HIF-1�, caspase-3, Epo and EpoR in the infarct,arginal and normal areas are represented in Figs. 6 and 7.In Groups 1–4, HIF-1� and caspase-3 expressions were observed

lmost only in the marginal area, while Epo and EpoR werexpressed in both the marginal and normal areas (Fig. 7A–D).

Caspase-3 expression in the marginal area was higher in Groups and 2 than Groups 3 and 4 (p = 0.055) (Fig. 6A). Epo administrationeduced caspase 3 expression in the marginal area, but there waso statistically significant difference (Figs. 6A and 7A). In Group 5,IF-1� and caspase-3 expression was not observed (Fig. 7A and B),nd there was only weak and ubiquitous Epo and EpoR expressionn all areas (Fig. 7C and D).

Epo and EpoR expressions were significantly more intense inhe marginal area than in the normal area (Figs. 6C, D and 7C, D).poR expression in the normal area was significantly more intensen Group 1 than the other groups (p < 0.05) (Figs. 6D and 7D). There


ere no significant differences in Epo expression in the infarct andarginal areas between the groups, however it was observed that

poR expression was significantly more intense in Group 1 than inroups 2 and 5 (p = 0.014) (Figs. 6D and 7D). EpoR expression was

lic in infarct areas (I). C–D; Marked vascularization (�) was seen in 6 Hour + EPOogical features were not observed. Scale bars: A = 500 �m; B = 200 �m; C = 100 �m;

significantly more intense in the marginal area in Groups 1, 3, and4, all expressing similar staining intensity (Figs. 6D and 7D).

Vascular Epo expression was significantly more intense inGroups 2 and 4 than Group 5 (p = 0.026) (Fig. 6C).

Discussion

The score of infarct area was found to be significantly lower inthe Epo-treated groups than the untreated groups, but there wasno significant difference between the treatment groups. Althoughthe duration of ischemia was longer in the Epo-treated groups thanGroup 1, the score of infarct was lower in these groups. The serumlevels of human Epo in rats were negatively correlated with thescore of infarct and hemorrhage. The findings observed by electronmicroscopy were less evident in the treatment groups. The resultssuggest that Epo administration during coronary artery occlusionsignificantly reduces the ischemic damage.

Cardioprotective effects of Epo administration were related tothe serum human Epo levels. It was considered that the levelsof serum human Epo representing the circulating fraction of Epoadministered intraperitoneally were an indicator of effective Epotreatment. The levels of serum human Epo were significantly higher


in the Epo-treated groups than others.We found that caspase-3 expression was located in the marginal

area surrounding the infarct. This marginal expression of caspase-3 indicates that the apoptotic activity involves the ischemic



ARTICLE ING Model


6 A. Guven Bagla et al. / Acta Histoc

Fig. 3. The stacked bars represent the frequency of different scores of infarction,hemorrhage and vascularization evaluated by light microscopy in each group. The“y” axis on the stacked bars represents rat numbers. Each part of the bars denoted bya different pattern shows the number of rats that have related score expressed in theright upper corner of each graphic. The differences between groups are statisticallysignificant. Letters over the bars (a, b, or c) denote a homogeneous subset of groupsthat is significantly different from each other at the 0.05 level in the multiple com-parisons. The groups denoted by the different letters are significantly different fromeach other (a is significantly different from both b and c, and also b is significantlydifferent from c), and the groups denoted by the same letter are statistically similar.The group denoted by two letters (a, b) is similar to both groups denoted by each ofthese letters (a, b is similar to both a and b, but a is still significantly different fromb). Results of A. the score of infarction (p < 0.001), B. hemorrhage (p = 0.002), and C.vascularization (p = 0.014).


myocardium surrounding the infarct area. There was a strongpositive correlation between marginal caspase-3 expression andboth the score of infarction and hemorrhage. Apoptotic activitythat occurred in the marginal area is responsible for worseningof the ischemic myocardial damage. It is well-known that Epoexerts its cardioprotective effects mainly through PI3K-Akt andJAK2-STAT pathways, which are responsible for inhibition of apo-ptosis (Calvillo et al., 2003; Moon et al., 2003; Parsa et al., 2003;Tramontano et al., 2003; Cai and Semenza, 2004; Lipsic et al., 2004;Wright et al., 2004; Bullard et al., 2005; Fiordaliso et al., 2005;Hanlon et al., 2005; Brines and Cerami, 2006; Mudalagiri et al.,2008; Ueba et al., 2010). We found a negative correlation betweenthe levels of serum human Epo and caspase-3 expression in themarginal area. Also, caspase-3 expression in the marginal areawas near-significantly lower in the treatment groups than Group 2(p = 0.055). It was concluded that reduced myocardial damage maybe associated with reduced apoptotic activity in the marginal area.

Ben-Dor et al. (2007) evaluated the protective potential of var-ious doses of Epo in a rat myocardial infarction model of leftventricular remodeling, specifically with regard to 6 weeks func-tion. In their study, Doppler echocardiography showed significantimprovement in LVFS (left ventricle fractional shortening) in thegroup treated with repeated low doses of Epo (750 U/kg) follow-ing coronary artery ligation, whereas it showed deterioration in theother groups (the group treated with a single dose Epo (5000 U/kg),the group treated with repeated high doses of Epo (1000 U/kg)and the no-treatment group). They suggested that these dose-dependent unexpected effects of Epo may reflect either systemicand myocardial effects or cumulative doses depending on fre-quency Epo administration. However, they did not find significantdifferences between Epo treatment groups and the control groupregarding the remodeling indices (end diastolic and end systolicareas, left ventricle circumference). They observed significantlyless collagen staining in non-infarct areas in single dose Epo andrepeated high doses Epo groups compared to no-treatment andrepeated low doses Epo groups. In addition, they reported that Epotreatment groups revealed reduction in apoptosis when comparedwith the no-treatment group (Ben-Dor et al., 2007). Similarly, weobserved that two different single doses of Epo reduced apopto-sis. High dose and standard dose Epo treatments revealed the sameresults.

Oxygen dependent regulation of Epo production is controlledby HIF-1 and -2 (Nangaku and Eckardt, 2007; Marzo et al., 2008).Both HIF-1 and -2 are composed of � and � subunits, and HIF-�/� heterodimers composed under hypoxic conditions induce Epotranscription (Marzo et al., 2008; Weidemann and Johnson, 2008).In our study, immunohistochemical expression of HIF-1� in theischemic myocardium was analyzed, and it was showed that HIF-1� expression occurred almost only in the marginal area likecaspase-3 expression. The results suggested that the major sourceof HIF-1� production induced by ischemic myocardial damage wasthe ischemic myocardium surrounding the infarct, but not theinfarcted myocardium itself. Also, HIF-1� expression was found tobe positively correlated with ischemia related variables includingthe score of infarction, hemorrhage, macrophage count, leukocyteincrease, and caspase-3 expression. It is unclear whether HIF is apro- or anti-apoptotic factor. It may induce caspase-3 expressionin ischemic myocardium. There is a strong correlation betweenHIF-1� and caspase-3 expression in the marginal area. On theother hand, it is known that Epo induced under hypoxic conditionsby HIF exerts important cardioprotective effects through reduc-ing apoptosis (Calvillo et al., 2003; Moon et al., 2003; Parsa et al.,


2003;Tramontano et al., 2003; Cai and Semenza, 2004; Lipsic et al.,2004; Wright et al., 2004; Bullard et al., 2005; Fiordaliso et al.,2005; Hanlon et al., 2005; Brines and Cerami, 2006; Mudalagiriet al., 2008; Ueba et al., 2010). Unlike caspase-3 and HIF-1�





Fig. 4. A. The bars represent the mean macrophage numbers in light microscopy. The difference between groups is statistically significant (p < 0.027). Letters over the barsdenote a homogeneous subset of groups (for a more thorough explanation, please refer to the legend of Fig. 3). B. The stacked bars represent the frequency of the scores ofneutrophil leukocyte increase in light microscopy (for a more thorough explanation, please refer to the legend of Fig. 3). The difference is statistically insignificant (p = 0.197).

Fig. 5. The images show the myocardium in electron microscopy (scale bars = 20 �m). A. Regular cardiomyocytes with mitochondria and myofilaments in control Group5 uoliza( lasmid ent d

eniemcEpEiitoEtA

. B. Mild degeneration and swelling of the mitochondria (m), intracytoplasmic vacarrowhead) in Group 1. C. Severe degeneration of the mitochondria (m), intracytopisks (arrowhead) in Group 2. D. Quite regular mitochondria (m), reduced myofilam

xpression, Epo expression was detected in both the marginal andormal areas. Although the strongest Epo expression was observed

n the marginal area, there was a weak or moderate degree Epoxpression in the normal area. Epo expression in the non-ischemicyocardium including either the normal area in the groups with

oronary ligation or the sham control group suggests ubiquitouspo production under normoxic conditions. This basal ubiquitousroduction of Epo is increased dramatically by ischemia or hypoxia.po was not expressed in the infarct area. Early Epo clearance andnability of transcriptional production of new Epo molecules in thenfarcted myocardium was attributed to lack of Epo expression inhe infarct area. Also, exogenous Epo administration did not induce


r reduce the endogenous Epo activity. There was no difference inpo expression between the groups, and no correlation betweenhe level of serum human Epo and the marginal Epo expression.dditionally, the level of serum rat Epo was similar in all groups

tion, and focal myofilament disarray (arrow), and fairly regular intercalated disksc vacuolization, severe myofilament disarray (arrow) and degenerated intercalatedisarray (arrow) and regular intercalated disks (arrowhead) in Group 3.

(p = 0.507). It was concluded that the cardioprotective effects of Epoadministration were independent of endogenous Epo activity, andexogenous Epo treatment did not suppress it.

Similarly, EpoR expression was observed in both the marginaland normal areas. Like Epo expression, the strongest EpoR expres-sion was observed in the ischemic marginal area. Also, there wasubiquitous EpoR expression in the non-ischemic myocardium.There was no or minimal EpoR expression in the infarct area. Lackof EpoR expression in the infarct area was similarly attributed toearly EpoR clearance and inability of transcriptional productionof new EpoR monomers in the infarcted myocardium. Moder-ate or strong EpoR expression in the marginal area suggested


that ischemia induces EpoR expression in the myocardium.Mechanisms related to EpoR expression in the ischemicmyocardium are unclear. We found that EpoR expression inthe marginal and normal area was significantly higher in the early




8 A. Guven Bagla et al. / Acta Histochemica xxx (2013) xxx– xxx

Fig. 6. The stacked bars represent the frequency of immunohistochemical expression of caspase-3, HIF-1�, Epo, and EpoR in the infarct, marginal, and normal areas accordingt the bae rs overp

poimmn

o the groups. The “y” axis on the stacked bars represents rat numbers. Each part ofxpressed in the right upper corner of the figure. p values are shown on figures. Lettelease refer to the legend of Fig. 3).

eriod of infarction (in Group 1). It was concluded that interactionf Epo/EpoR may cause the consumption of EpoR homodimers


n the normal area. Unlike Epo expression, there is no knownediator of EpoR expression. Local factors affected by ischemiaay induce EpoR expression. Lack of ischemic stimulus in the

ormal area and consumption of EpoR molecules by Epo/EpoR

rs denoted by a different pattern shows the number of rats that have related score the bars denote a homogeneous subset of groups (for a more thorough explanation,

interaction may cause a reduction in EpoR expression with time.However, EpoR production in the ischemic marginal area still


remains. It is unclear whether exogenous Epo administrationcontributes to the consumption of EpoR, because the treatmentgroups were not sacrificed during the early period of ischemia. Inthe later period of ischemia, exogenous Epo administration was





Fig. 7. Representative micrographs of immunohistochemical expression of caspase-3, HIF-1�, Epo, and EpoR: A. Caspase 3 expression: Caspase 3 expression was not observedin the infarcted area (I) and normal area (N) in working groups. Caspase 3 expression was observed only in marginal area (M). B. HIF-1� expression: HIF-1� expression wasnot observed in the infarcted area (I) and normal area (N) in working groups. HIF-1� expression was observed only in marginal area (M). In the control group, HIF-1�expression was not observed. C. Epo expression: Epo expression was not observed in the infarcted areas (I). In the marginal areas (M) of working groups, Epo expression wasm s diffuw t obsee ressio

ntGmp

iiAlncintttNif(tsbgaiEfn

ivcnba

ore intense than that of the normal areas (N). In control group, Epo expression waas the most intensely expressed in the marginal area (M). EpoR expression was no

xpressed less intensely than that of marginal areas. In the control group, EpoR exp

ot found to be associated with impaired EpoR expression, becausehe degree of EpoR expression in the normal area was similar inroups 2–4. It should be further explored whether a mediatoray induce EpoR expression in the ischemic myocardium like Epo

roduction.Enhancing new vessel formation over a longer time frame

s one of the major cardioprotective effects of Epo againstschemia/reperfusion injury (Parsa et al., 2003; Lipsic et al., 2006).lthough there was a strong positive correlation between vascu-

arization and the levels of serum human Epo, vascularization wasot correlated with the score of infarction and hemorrhage. It wasoncluded that Epo administration induces new vessel formationn the ischemic myocardium during the first 6 h of infarction, buteovascularization does not contribute to the myocardial protec-ion during this early period of infarction. Neovascularization ofhe ischemic myocardium is associated with long-term cardiopro-ective effects of Epo (Lipsic et al., 2006). It was demonstrated byakano et al. (2007) that the vascular Epo/EpoR system promotes

schemia-induced neovascularization. Vascular Epo expression wasound to be positively correlated with vascularization in our studyp = 0.004). Also, vascular Epo expression had a positive correla-ion with the levels of serum human Epo (p = 0.006) (data nothown). Nakano et al. (2007) related neovascularization promotedy local vascular Epo and EpoR to enhanced vascular endothelialrowth factor secretion, endothelial progenitor cells mobilization,nd recruitment of bone marrow derived proangiogenic cells to theschemic tissue (Nakano et al., 2007; Brunner et al., 2012). Vascularpo expression could be an important mechanism in new vesselormation during ischemia, and Epo administration may induceeovascularization via improving vascular Epo expression.

In conclusion: (i) Epo treatment reduces the apoptotic activityn the ischemic myocardial damage in rats. (ii) It also induces newessel formation in the early period of infarction, but this neovas-


ularization does not contribute to myocardial protection. There iso significant correlation between the score of vascularization andoth the infarct size (r = 0.175 and p = 0.426) and score of infarctrea (r = −0.317 and p = 0.06). We conclude from this result that

sely weak. D. EpoR expression: 1 h after coronary artery ligation group (1 h): EpoRrved in the infarcted area (I). In the normal areas (N) of working groups, EpoR wasn was diffusely weak. Scale bars = 50 �m.

vascularization that occurred during the early period of infarctiondoes not provide additional myocardial protection. (iii) The cardio-protective effects of Epo treatment is independent of endogenousEpo/EpoR activity, and exogenous Epo treatment does not suppressit. Epo shows its cardioprotective effects through its receptor, EpoR.It was thought that Epo and EpoR form a unit working together.For that reason, we defined the Epo and EpoR unit as Epo/EpoR.Actually, from this we cannot conclude that the cardioprotectiveeffects of Epo are independent of endogenous EpoR activity as welack the data to reach such a conclusion. The term “endogenousEpo/EpoR activity” only represents the functional collaborationbetween these two molecules. (iv) Higher dose of Epo administra-tion does not provide additional benefit beyond a standard-dose inmyocardial protection, because higher dose of intraperitoneal Epoinjection do not elevate the levels of serum human Epo more thana standard-dose injection. More studies need to be undertaken toexplain the relationship between acute myocardial infarction andthe cardioprotective effects of different erythropoietin doses.

Acknowledgement

This study was supported in part by Scientific Research ProjectCommission of C anakkale Onsekiz Mart University [grant number2010/122].

References

Ahmet I, Tae HJ, Juhaszova M, Riordon DR, Boheler KR,Sollott SJ, et al. A small nonerythropoietic helix B surfacepeptide based upon erythropoietin structure is cardioprotec-tive against ischemic myocardial damage. Mol Med 2011;17:194–200.


Akimoto T, Kusano E, Inaba T, Iimura O, Takahashi H, Ikeda H, et al.Erythropoietin regulates vascular smooth muscle cell apoptosisby a phosphatidylinositol 3 kinase-dependent pathway. KidneyInt 2000;58:269–82.


ING Model

A

1 Histoc

A

A

A

B

B

B

B

B

C

C

C

C

D

D

F

F

G


0 A. Guven Bagla et al. / Acta

mmarguellat F, Llovera M, Kelly PA, Goffin V. Low doses ofEPO activate MAP kinases but not JAK2-STAT5 in rat vascularsmooth muscle cells. Biochem Biophys Res Commun 2001;284:1031–8.

nagnostou A, Liu Z, Steiner M, Chin K, Lee ES, Kessimian N, et al.Erythropoietin receptor mRNA expression in human endothelialcells. Proc Natl Acad Sci USA 1994;91:3974–8.

zevedo Filho CF, Hadlich M, Petriz JL, Mendonc a LA, Moll FilhoJN, Rochitte CE. Quantification of left ventricular infarcted masson cardiac magnetic resonance imaging, Comparison betweenplanimetry and the semiquantitative visual scoring Method. ArqBras Cardiol 2004;83:118–24, 111–117.

en-Dor I, Hardy B, Fuchs S, Kaganovsky E, Kadmon E, SagieA, et al. Repeated low-dose of erythropoietin is associ-ated with improved left ventricular function in rat acutemyocardial infarction model. Cardiovasc Drugs Ther 2007;21:339–46.

rines M, Cerami A. Discovering erythropoietin’s extra-hematopoietic functions: biology and clinical promise. KidneyInt 2006;70:246–50.

rines M, Grasso G, Fiordaliso F, Sfacteria A, Ghezzi P, FratelliM, et al. Erythropoietin mediates tissue protection through anerythropoietin and common beta-subunit heteroreceptor. ProcNatl Acad Sci USA 2004;101:14907–12.

runner S, Huber BC, Weinberger T, Vallaster M, WollenweberT, Gerbitz A, et al. Migration of bone marrow-derived cellsand improved perfusion after treatment with erythropoietinin a murine model of myocardial infarction. J Cell Mol Med2012;16:152–9.

ullard AJ, Govewalla P, Yellon DM. Erythropoietin protects themyocardium against reperfusion injury in vitro and in vivo. BasicRes Cardiol 2005;100:397–403.

ai Z, Manalo DJ, Wei G, Rodriguez ER, Fox-Talbot K, Lu H, et al.Hearts from rodents exposed to intermittent hypoxia or eryth-ropoietin are protected against ischemia-reperfusion injury.Circulation 2003;108:79–85.

ai Z, Semenza GL. Phosphatidylinositol-3-kinase signalingis required for erythropoietin-mediated acute protectionagainst myocardial ischemia/reperfusion injury. Circulation2004;109:2050–3.

alvillo L, Latini R, Kajstura J, Leri A, Anversa P, GhezziP, et al. Recombinant human erythropoietin protects themyocardium from ischemia-reperfusion injury and promotesbeneficial remodeling. Proc Natl Acad Sci USA 2003;100:4802–6.

hong ZZ, Kang JQ, Maiese K. Hematopoietic factor erythropoie-tin fosters neuroprotection through novel signal transductioncascades. J Cereb Blood Flow Metab 2002;22:503–14.

igicaylioglu M, Bichet S, Marti HH, Wenger RH, Rivas LA, BauerC, et al. Localization of specific erythropoietin binding sitesin defined areas of the mouse brain. Proc Natl Acad Sci USA1995;92:3717–20.

igicaylioglu M, Lipton SA. Erythropoietin-mediated neuroprotec-tion involves cross-talk between Jak2 and NF-kappaB signallingcascades. Nature 2001;412:641–7.

iordaliso F, Chimenti S, Staszewsky L, Bai A, Carlo E, CuccovilloI, et al. A nonerythropoietic derivative of erythropoietin pro-tects the myocardium from ischemia-reperfusion injury. ProcNatl Acad Sci USA 2005;102:2046–51.

isher JW, Koury S, Ducey T, Mendel S. Erythropoietin productionby interstitial cells of hypoxic monkey kidneys. Br J Haematol1996;95:27–32.


uneli E, Cavdar Z, Islekel H, Sarioglu S, Erbayraktar S,Kiray M, et al. Erythropoietin protects the intestine againstischemia/reperfusion injury in rats. Mol Med 2007;13:509–17.


Hanlon PR, Fu P, Wright GL, Steenbergen C, Arcasoy MO,Murphy E. Mechanisms of erythropoietin-mediated cardio-protection during ischemia-reperfusion injury: role of proteinkinase C and phosphatidylinositol 3-kinase signaling. FASEB J2005;19:1323–5.

Jelkmann W, Wagner K. Beneficial and ominous aspects ofthe pleiotropic action of erythropoietin. Ann Hematol2004;83:673–86.

Jelkmann W. Molecular biology of erythropoietin. Intern Med2004;43:649–59.

Junk AK, Mammis A, Savitz SI, Singh M, Roth S, Malhotra S,et al. Erythropoietin administration protects retinal neuronsfrom acute ischemia-reperfusion injury. Proc Natl Acad Sci USA2002;99:10659–64.

Lipsic E, Schoemaker RG, van der Meer P, Voors AA,van Veldhuisen DJ, van Gilst WH. Protective effects of erythro-poietin in cardiac ischemia: from bench to bedside. J Am CollCardiol 2006;48:2161–7.

Lipsic E, van der Meer P, Henning RH, Suurmeijer AJ, Boddeus KM,van Veldhuisen DJ, et al. Timing of erythropoietin treatment forcardioprotection in ischemia/reperfusion. J Cardiovasc Pharma-col 2004;44:473–9.

Marti HH. Erythropoietin and the hypoxic brain. J Exp Biol2004;207:3233–42.

Marzo F, Lavorgna A, Coluzzi G, Santucci E, Tarantino F, Rio T, et al.Erythropoietin in heart and vessels: focus on transcription andsignalling pathways. J Thromb Thrombolysis 2008;26:183–7.

Moon C, Krawczyk M, Ahn D, Ahmet I, Paik D, Lakatta EG, et al.Erythropoietin reduces myocardial infarction and left ventricu-lar functional decline after coronary artery ligation in rats. ProcNatl Acad Sci USA 2003;100:11612–7.

Mudalagiri NR, Mocanu MM, Di Salvo C, Kolvekar S, Hayward M, YapJ, et al. Erythropoietin protects the human myocardium againsthypoxia/reoxygenation injury via phosphatidylinositol-3 kinaseand ERK1/2 activation. Br J Pharmacol 2008;153:50–6.

Nakano M, Satoh K, Fukumoto Y, Ito Y, Kagaya Y, Ishii N, et al.Important role of erythropoietin receptor to promote VEGFexpression and angiogenesis in peripheral ischemia in mice. CircRes 2007;100:662–9.

Nangaku M, Eckardt KU. Hypoxia and the HIF system in kidneydisease. J Mol Med 2007;85:1325–30.

Ogilvie M, Yu X, Nicolas-Metral V, Pulido SM, Liu C, Ruegg UT, et al.Erythropoietin stimulates proliferation and interferes with dif-ferentiation of myoblasts. J Biol Chem 2000;275:39754–61.

Parsa CJ, Matsumoto A, Kim J, Riel RU, Pascal LS, Walton GB, et al. Anovel protective effect of erythropoietin in the infarcted heart.J Clin Invest 2003;112:999–1007.

Paschos N, Lykissas MG, Beris AE. The role of erythropoietinas an inhibitor of tissue ischemia. Int J Biol Sci 2008;4:161–8.

Rui T, Feng Q, Lei M, Peng T, Zhang J, Xu M, et al. Erythropoietinprevents the acute myocardial inflammatory response inducedby ischemia/reperfusion via induction of AP-1. Cardiovasc Res2005;65:719–27.

Sasaki R, Masuda S, Nagao M. Erythropoietin: multiple physiolog-ical functions and regulation of biosynthesis. Biosci BiotechnolBiochem 2000;64:1775–93.

Sharples EJ, Patel N, Brown P, Stewart K, Mota-Philipe H, Sheaff M,et al. Erythropoietin protects the kidney against the injury anddysfunction caused by ischemia-reperfusion. J Am Soc Nephrol2004;15:2115–24.

Solaroglu A, Dede FS, Okutan E, Bayrak A, Haberal A, Kilinc


K. A single dose of erythropoietin attenuates lipid per-oxidation in experimental liver ischemia-reperfusion injuryin the rat fetus. J Matern Fetal Neonatal Med 2004;16:231–4.


ING Model

A

Histoc

T

U

V



ramontano AF, Muniyappa R, Black AD, Blendea MC, Cohen I,Deng L, et al. Erythropoietin protects cardiac myocytes fromhypoxia-induced apoptosis through an Akt-dependent path-way. Biochem Biophys Res Commun 2003;308:990–4.

eba H, Brines M, Yamin M, Umemoto T, Ako J, Momomura S, et al.Cardioprotection by a nonerythropoietic, tissue-protective pep-tide mimicking the 3D structure of erythropoietin. Proc Natl


Acad Sci USA 2010;107:14357–62.ivaldi MT, Kloner RA, Schoen FJ. Triphenyltetrazolium staining of

irreversible ischemic injury following coronary artery occlusionin rats. Am J Pathol 1985;121:522–30.

PRESShemica xxx (2013) xxx– xxx 11

Weidemann A, Johnson RS. Biology of HIF-1 alpha. Cell Death Differ2008;15:621–7.

Wright GL, Hanlon P, Amin K, Steenbergen C, Murphy E, ArcasoyMO. Erythropoietin receptor expression in adult rat cardiomy-ocytes is associated with an acute cardioprotective effectfor recombinant erythropoietin during ischemia-reperfusioninjury. FASEB J 2004;18:1031–3.


Wu H, Ren B, Zhu J, Dong G, Xu B, Wang C, et al. Pretreatmentwith recombined human erythropoietin attenuates ischemia-reperfusion-induced lung injury in rats. Eur J Cardiothorac Surg2006;29:902–7.


Documents

Experimental acute myocardial infarction in rats: HIF-1α, caspase-3, erythropoietin and erythropoietin receptor expression and the cardioprotective effects of two different erythropoietin