The protective effects of tacrolimuson rat uteri exposed toischemia-reperfusion injury: abiochemical andhistopathologic evaluation

Sadik Sahin, M.D.,a Ozlem Bingol Ozakpinar, Ph.D.,b Koray Ak, M.D.,c Mustafa Eroglu, M.D.,a

Merve Acikel, M.Sc.,d Sermin Tetik, Ph.D.,b Fikriye Uras, Ph.D.,b and Sule Cetinel, M.D.d

a Department of Obstetrics and Gynecology, Zeynep Kamil Gynecologic and Pediatric Training and Research Hospital;b Department of Biochemistry, School of Pharmacy, and c Department of Cardiovascular Surgery and d Department ofHistology and Embryology, School of Medicine, Marmara University, Istanbul, Turkey

Objective: To evaluate the effects of the immunosuppressant tacrolimus as an antioxidant and analyze the histopathologic changes inrat uteri exposed to experimental ischemia-reperfusion (I/R) injury.Design: Experimental study.Setting: Experimental surgery laboratory in a university.Animal(s): Twenty-eight female rats exposed to experimentally induced uterine I/R injury.Intervention(s): Group I: control group; group II: uterine I/R injury–induced group; group III: pre-ischemia tacrolimus group; group IV:post-ischemia tacrolimus group.Main OutcomeMeasure(s): Uterine tissue malondialdehyde (MDA) level as a marker of lipid peroxidation and glutathione (GSH) leveland superoxide dismutase (SOD) and catalase (CAT) activities as markers of tissue antioxidant capacity; histopathologic examination ofall uterine rat tissue.Result(s): Following aortic I/R injury, MDA levels were significantly increased whereas GSH levels and CAT and SOD activities werefound to be decreased compared with control animals. MDA levels were found to recover prominently after the administration of ta-crolimus in both groups III and IV. Administration of tacrolimus improved uterine GSH levels and CAT activity in the tacrolimus-treatedgroups.Conclusion(s): Our results indicate that tacrolimus reduces oxidative damage in rat uteri exposed to I/R injury induced by distal

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abdominal aortic occlusion. Histologic evaluation reveals that tacrolimus attenuates the inflam-matory response and protects the tissue damage induced by I/R injury. (Fertil Steril� 2014;101:1176–82. �2014 by American Society for Reproductive Medicine.)Key Words: Ischemia-reperfusion injury, tacrolimus, uterus, rat, transplantation

Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/sahins-tacrolimus-rat-uterus-ischemia-reperfusion-injury/

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terine transplantation (UT) has few years. Patients with uncorrectable include those with the congenital

U been gaining popularityaround the world in the past

Received September 27, 2013; revised December 25online February 4, 2014.

S.S. has nothing to disclose. O.B.O. has nothing tonothing to disclose. M.A. has nothing to disclosto disclose. S.C. has nothing to disclose.

Reprint requests: Sadik Sahin, M.D., Zeynep Kamil GHospital, Op. Dr. Burhanettin Ustunel Cd No:10gmail.com).

Fertility and Sterility® Vol. 101, No. 4, April 2014 00Copyright ©2014 American Society for Reproductivehttp://dx.doi.org/10.1016/j.fertnstert.2013.12.044

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uterine-factor infertility are consideredto be ideal candidates for UT and

, 2013; accepted December 26, 2013; published

disclose. K.A. has nothing to disclose. M.E. hase. S.T. has nothing to disclose. F.U. has nothing

ynecologic and Pediatric Training and Research, 34668 Istanbul, Turkey (E-mail: drsadiksahin@

15-0282/$36.00Medicine, Published by Elsevier Inc.

absence of the uterus (Mayer-Rokitan-sky-K€uster-Hauser syndrome) and pa-tients hysterectomized owing tobenign conditions, malignant tumors,or massive blood loss after delivery.Furthermore, patients with nonfunc-tional uterine cavities are also candi-dates for UT (1). Currently, options forpatients with uterine-factor infertilityare gestational surrogacy or adoption.However, surrogacy is legal in only afew countries, owing to concerns about

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ethical, social, and legal issues (2). In light of current studies,UT may become the first treatment option for women withuterine-factor infertility in these and other countries. The firstattempt of UT in humans was carried out in Saudi Arabia in2000 (3). Approximately 3 months after the transplantation,the uterus had to be removed owing to uterine prolapse andnecrosis. It was thought that this attempt of human uterinetransplantation had been made without sufficient animalresearch. Since that first attempt, many studies have beenconducted in animals. Successful transplantation and preg-nancy has been achieved in mice, sheep, and rats (4–6).Based on data accumulated from animal studies, the secondattempt of UT in humans was conducted from a cadaverdonor in Turkey in 2011. Although it was technicallysuccessful, it could not produce an uneventful pregnancywith birth of a healthy baby (7). Following this, a Swedishgroup performed 2 procedures, which were the world’s firstliving-donor UTs from mothers to their daughters (8).

Ischemia-reperfusion (I/R) injury has detrimental effects ontransplanted organs (9). In general, I/R injury is mediated byseveral mechanisms, including the release of reactive oxygenspecies (ROS), activation of leukocytes, endothelial system dys-functions and activation of complement pathways (10, 11).ROS are one of the most important components of I/R injurycausing apoptotic and necrotic cell death via lipid peroxidationin a variety of organs (12). Lipid peroxidation is a catalyticmechanism leading to oxidative destruction of cellularmembranes. Therefore, lipid peroxidation has been suggestedto be closely related to I/R injury–induced tissue damage, andmalondialdehyde (MDA) is a good indicator of the rate of lipidperoxidation (13). Endogenous antioxidant enzymes, such assuperoxide dismutase (SOD), catalase (CAT), and glutathione(GSH), protect cells from the detrimental effects of ROS. Thelevels of antioxidant enzymes have been used to indicate themagnitude of oxidative stress that occurs during I/R injury (14).

Tacrolimus, a calcineurin inhibitor, is a crucial immuno-suppressant used after kidney, liver, and pancreas transplan-tations (15–17). It has diverse actions that result in thereduction of I/R injury. Several investigators havedocumented decreased ROS production in association with areduction in I/R injury when tacrolimus is administeredbefore ischemia (18, 19). Endothelial damage triggersinteractions among platelets, leukocytes, and endothelialcells, thus leading to the increased secretion of cytokinesand infiltration of mononuclear cells. Tacrolimus attenuatesthis cascade by inhibiting the secretion of ROS and reducingthe expression of adhesion molecules including P-selectinand intercellular adhesion molecule 1 (18, 20).

The aim of the present study was to examine the effects oftacrolimus on uterine I/R injury induced by distal abdominalaortic occlusion. For this purpose, the rat uterine tissue levelsof MDA, GSH, SOD, and CAT were measured. Furthermore,the rat uterine specimens were histologically evaluated.

MATERIALS AND METHODSAnimals

A total of 28 female Wistar albino rats (3–4 months old)weighing 230–270 g obtained from the Marmara University

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Experimental Animal Research Laboratory were enrolled inthe study. They were given free access to water and standardlaboratory rodent pellet food. The rats were maintained in thelaboratory under controlled environmental conditions (roomtemperature 22 � 1�C) and 12-hour:12-hour light-dark cy-cles. All rats were used in compliance with the national guide-lines for the use and care of laboratory animals. The protocolswere designed according to the Committee of Ethics on Ani-mal Experimentation and approved by the Institutional Re-view Board of Marmara University (report no. 83.2013.mar.).

Chemicals

Tacrolimus (Prograf, 5 mg/mL), ketamine hydrochloride (Ke-talar, 50 mg/mL), and xylazine (Rompun, 20 mg/mL) werepurchased from Astellas, Pfizer, and Bayer, respectively.

Experimental Groups and Surgical Technique

The rats were randomized into four groups of seven rats each.All rats included in the study were synchronized in the dies-trus phase of estrous cycle, which was judged as describedby Marcondes et al. (21). Group I (the control group) consistedof rats that did not receive any treatment, group II (the uterineI/R injury group) of rats exposed to 0.5 hour of ischemia and 1hour of reperfusion, group III (the pre-ischemia tacrolimusgroup) of rats that received intravenous tacrolimus (0.3 mg/kg) 0.5 hour before the induction of I/R, and group IV (thepost-ischemia tacrolimus group) of rats that received tacroli-mus (0.3 mg/kg) immediately before reperfusion. In groups IIIand IV, the given dosage of tacrolimus (0.3 mg/kg) wasdecided according to that reported to be protective in liverwarm ischemia reperfusion injury model (22).

The abdominal aorta in the control group was dissectedunder laparotomy but was not occluded. The other threegroups were exposed to ischemia and reperfusion by occlu-sion of the distal abdominal aorta and collateral occlusionof the ovarian arterial blood supply below the level of theovaries.

The rats were anesthetized with a combination of keta-mine hydrochloride (60 mg/kg intraperitoneally) and xyla-zine (5 mg/kg intraperitoneally), and supplementaryinjections of ketamine hydrochloride were applied as needed.When the anesthesia was accomplished, the rats were placedin a supine position and a midline laparotomy was carried outunder aseptic conditions. The abdominal aorta was exposedby gently deflecting the loops of intestine to the left sidewith moist gauze swabs. An atraumatic microvascular clamp(Bulldog clamp; Aesculap) was placed across the distalabdominal aorta just above the bifurcation of iliac arteries,and two more vascular clamps were applied below bothovaries to prevent collateral blood supply. After this, theabdomen was closed and the wound was covered with moistgauze to minimize heat and fluid loss. After a 30-minuteischemia period, all clamps were removed and reperfusionwas allowed for 60 minutes. All rats were then killed underanesthesia and both uterine horns were carefully removed.The left uterine horns were reserved for biochemical assaysand stored at �80�C until analysis. The right uterine horns

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were transferred into a 10% formaldehyde and 4% glutaralde-hyde solution for histopathologic examination.

Biochemical Investigations

Uterine MDA and GSH levels. Uterine tissue samples werehomogenized with ice-cold 150 mmol/L KCl for biochemicalanalyses. MDA levels were assessed as an indicator of lipidperoxidation with the use of the method defined by Okhawaet al (23). Lipid peroxide levels were expressed in terms ofMDA equivalents with the use of an extinction coefficientof 1.56 � 105 mol�1 L�1 cm�1 and the results were expressedas nmol MDA/g tissue. GSH was determined spectrophoto-metrically with the use of Ellman reagent (24). GSH levelswere calculated using an extinction coefficient of 1.36 �104 mol�1 L�1 cm�1, and results were expressed as mmolGSH/g tissue.

SOD and CAT activity. SOD activity was measured with theuse of the commercially supplied Superoxide DismutaseAssay Kit (Cayman Chemical) according to themanufacturer’sinstructions. The calculated SOD activity was expressed asU/g protein in uterine tissue samples. CAT and hydrogenperoxide (H2O2) scavenger levels were also determined ac-cording to the instructions for the kit from Cayman Chemical.The enzymatic activity was expressed as nmol min�1 mL�1.

Histologic Examination

For histopathologic analysis, uterine samples were fixed in10% buffered formalin for 48 hours, dehydrated in anascending alcohol series, and embedded in paraffin. Sections,�5 mm in thickness, were stained with hemotoxylin-eosin.Furthermore, the specimens were fixed in 4% glutaraldehyde(0.13 mol/L and pH 7.4) in phosphate-buffered saline solutionfor 4 hours and post-fixed with OsO4 for 1 hour. After this, thespecimens were dehydrated in a graded alcohol series andembedded in Epon 812. Semithin (1 mm) sections were stainedwith toluidine blue. Histologic assessments were performedwith the use of a photomicroscope (Olympus BX 51) by anexperienced histologist who was blinded to the experimentalgroups, and histologic alterations were graded semiquantita-tively as minimal, moderate, and severe.

Statistical Analysis

Statistical analyses were performed with the use of the Graph-pad Prism 6.0 software by one-way analysis of variance fol-lowed by Tukey post hoc analysis. Data were expressed asmean � SD, and differences between groups were consideredto be statistically significant at a P value of < .05.

RESULTSResults of Biochemical Investigations

The levels of MDA, a marker of lipid peroxidation, weremeasured as an indicator of oxidative injury to determineuterine damage. MDA levels in the I/R injury group (71.36� 9.91 nmol/g tissue) were significantly higher comparedwith the control group (38.08 � 6.39 nmol/g tissue;P< .0001; Fig. 1). MDA levels were significantly lower in

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both the pre-ischemia and the post-ischemia tacrolimusgroups (30.56 � 7.4 and 47.16 � 6.03 nmol/g tissue, respec-tively) compared with the I/R injury group (P< .0001 andP< .001, respectively). On the other hand, the MDA levels inthe post-ischemia tacrolimus group were significantly higherthan the pre-ischemia group (P< .01).

GSH is the major ROS scavenger in many tissues and or-gans. In the present study, uterine GSH levels in the I/R injurygroup (1.09 � 0.63 mmol/g tissue) were found to be signifi-cantly lower than in the control group (3.01 � 0.44 mmol/gtissue; P< .0001; Fig. 1). However, the groups treated with ta-crolimus (group III 3.24 � 0.29 mmol/g tissue and group IV2.48 � 8.4 mmol/g tissue) showed significant improvementin GSH levels compared with the I/R injury group (P< .0001and P< .001, respectively). Additionally, uterine GSH contentin the pre-ischemia tacrolimus group was significantly higherthan in the post-ischemia group (P< .05).

CAT activity in the I/R injury group was found to bereduced significantly compared with the control group (con-trol group 44.98 � 8.75 nmol min�1 mL�1 and I/R injurygroup 20.08 � 8.62 nmol min�1 mL�1; P< .0001; Fig. 1). Incontrast, in the tacrolimus-treated pre- and post-ischemiagroups, CAT activity was increased significantly (37.68 �5.49 and 29.43 � 4.19 nmol min�1 mL�1, respectively)compared with group II (P< .0001 and P< .05, respectively).Furthermore, CAT activity in the pre-ischemia group washigher than in the post-ischemia group (P< .05).

Tissue SOD levels were found to be decreased signifi-cantly following the induction of distal abdominal aortic I/R injury (control group 38.86� 7.01 U/g tissue and I/R injurygroup: 23.63 � 63.16 U/g tissue; P< .01; Fig. 1). Administra-tion of tacrolimus in groups III and IV led to the elevation ofSOD levels (34.16� 5.48 and 33.14� 7.78 U/g tissue, respec-tively) compared with group II, but this elevation was not sta-tistically significant (P>.05).

Results of Histologic Investigations

The control group displayed regular endometrial contours andglands (Fig. 2A), whereas the endometria of the I/R groupshowed a severe accumulation of polymorphonuclear (PMN)cells and disruption of glandular cells (Fig. 2B). Tacrolimustreatment before the induction of ischemia reduced the num-ber of PMN cells, and there was a prominent regeneration ofglandular cells (Fig. 2C). A regenerated morphology of laminapropria and glandular structures was also evident in the post-ischemia tacrolimus group, similarly to that of the pre-ischemia tacrolimus group (Fig. 2D). When the two treatmentgroups were compared, the group treated with tacrolimusbefore induction of ischemia revealed a fine contour of endo-metrial and glandular surface epithelium besides less glan-dular epithelial picnotic nuclei.

Examination of semithin sections displayed a moredetailed morphology of uterine tissue (Fig. 3). Control tissuesections demonstrated regular glandular structure and con-nective tissue (Fig. 3A). On the other hand, I/R injury in groupII led to severe degenerative changes in glands and connectivetissue (Fig. 3B). Glandular epithelial cells showed cytoplasmichypertrophy, basal disruption, and pyknosis. Moreover, the

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FIGURE 1

The levels of malondialdehyde (MDA), glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) in the control, ischemia-reperfusion (I/R),and tacrolimus-treated groups. Each group consists of seven rats. Values are expressed as mean � SD. *P<.0001, gP<.01 compared with controlgroup. **P<.0001, ***P<.001, þþP<.05 compared with I/R group. þP<.01, pP<.05 compared with pre-ischemia group.Sahin. The effects of tacrolimus on rat uteri. Fertil Steril 2014.

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lamina propria were prominently edematous. In the pre-ischemia tacrolimus group (Fig. 3C) the glandular epithelialcells displayed cytoplasmic regeneration, and the laminapropria were less edematous. The post-ischemia tacrolimusgroup (Fig. 3D) also displayed regenerative changes in glandsand lamina propria, though to a lesser degree compared withthe pre-ischemia group. Semiquantitative scoring of degener-ation in uterine tissue is assessed in Table 1. The degrees ofdegenerative changes were seen to a lesser degree in thepre- and post-ischemia tacrolimus groups compared withthe I/R injury group. Group III seemed to be the most protectedfrom the effects of I/R injury–induced degenerativealterations.

DISCUSSIONThis study revealed that tacrolimus had protective effects onuterine I/R injury in both of the treatment groups; this effectwas more marked in the pre-ischemia tacrolimus group thanin the post-ischemia tacrolimus group. In both of thetacrolimus-treated groups, tissue MDA levels were signifi-cantly decreased and levels of GSH and CAT activityincreased compared with the I/R group. Furthermore, tacroli-mus significantly attenuated the histopathologic changesassociated with aortic I/R-induced uterine injury.

Accumulating evidence reveals that I/R injury increasesthe rates of graft losses in solid organ transplantation (25).I/R injury is especially important in organs harvested fromdeceased donors, because both warm (between vessel clampingand cold perfusion) and cold ischemia (from flushing with coldperfusion solution to anastomosis) times may be prolonged in

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these cases (9). Warm ischemia becomes an important issue inuterus either retrieved from cardiac death donors or that mayhave vascular complications during the anastomosis of vesselsto the recipient. Diaz-Garcia et al (26) tested the effect of warmischemia on survival of rat uterus after transplantation andconcluded that extended longer warm ischemia times (240 mi-nutes) result inmore severe ischemic alterations and necrosis intransplanted uterine tissues than in the control group. Preser-vation of harvested organs from a deceased donor is anotherimportant issue because the transfer of organs to a recipientmay take a longer time, which differs from that in living donortransplantation. Current evidence suggests that cold preserva-tion solutions, such as heparinized Ringer acetate in a wellperfused uterus in pigs and a more complex solution, namely,Perfadex, in the sheep model, lessen oxidative stress andinflammation in the transplanted uterus tissue (27, 28). Inaccordance with these results, cold preservation solutions,such as University of Wisconsin and Perfadex, have beenshown to preserve the human uterine myometrial tissuethrough preserving adenosine triphosphate (ATP) stores afterat least 6 hours of cold ischemia (29).

The major consequence of I/R injury is oxidative stressleading to the generation of ROS. Mitochondrial respirationis an important source of ROS and thus a potential contributorto reperfusion injury. ROS may disturb the balance of thecellular oxidative status and lead to the destruction of cellmembranes by inducing lipid peroxidation (13). In the presentstudy, MDA levels increased significantly in the I/R injurygroup compared with the control group, whereas tacrolimussignificantly decreased the levels of MDA in the pre- andpost-ischemia treatment groups. These findings indicate that

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FIGURE 2

Light microscopy findings of rat uterine tissue. (A) Control group; regular alignment of endometrial epithelium (arrow) and glandular cells(arrowheads), some polymorphonuclear cells in lamina propria (*). (B) Ischemia-reperfusion group: severe accumulation of polymorphonuclearcells in lamina propria (*), congestion of vessels, disruption of cells in glandular epithelium (inset, arrows). (C) Pre-ischemia tacrolimus: reduceddensity of polymorphonuclear cells in lamina propria, regeneration of glandular cell morphology (arrows). (D) Post-ischemia tacrolimus:regeneration of both endometrial (inset, arrow) and glandular (arrow) epithelium, note the reduced population of polymorphonuclear cells (*).Staining: hematoxylin-eosin.Sahin. The effects of tacrolimus on rat uteri. Fertil Steril 2014.

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tacrolimus decreases the magnitude of oxidative stress andsubsequently lipid peroxidation. Consistent with our findings,Suzuki et al found that tacrolimus decreased the tissue levels ofMDA in tacrolimus-treated rats exposed to I/R injury of theliver (30). Perra et al also reported that tacrolimus-treated renaltransplant patients showed a significant decrease in plasmaMDA levels 6 months after the transplantation (31).

GSH, a well known strong antioxidant found in all cells,provides protection against oxidative stress by participatingin the cellular defense system against ROS (32). There is agood deal of evidence indicating that GSH acts as a cosub-strate for glutathione peroxidase (GPx), reducing intracellu-larly generated peroxides and acting as a direct radicalscavenger as well (33). In the present study, tissue GSH levelswere found to be decreased following I/R injury. On the otherhand, treatment with tacrolimus prevented GSH depletionsignificantly in both groups, especially in the pre-ischemiatacrolimus-treated group. In agreement with our findings,Pratschke et al reported that preconditioning with tacrolimusdecreased hepatocellular oxidative damage through participi-tation of GSH after liver transplantation (19). In anotherstudy, preadministration of tacrolimus in a myocardial I/Rinjury increased the GSH metabolism, which in turn may pro-tect organ function by reducing ROS toxicity (34). Based onthese reports, the decrease in tissue GSH levels may be due

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to its consumption during oxidative stress induced by uterineI/R injury and tacrolimus may prevent the depletion ofcellular GSH concentration.

Catalase is an antioxidant enzyme especially concen-trated in the liver. It converts H2O2 to H2O and O2, and this re-action prevents the formation of the highly reactive hydroxylradical OH� (33). Earlier studies showed that catalase mightprotect tissues and organs exposed to I/R injury by enhancingthe antioxidative ability and reducing oxidative stress (35,36). In parallel with these results, the levels of catalasedecreased significantly in the I/R injury group comparedwith the control group. In contrast, the tacrolimus-treatedgroups had significantly increased levels of CAT.

Calcineurin inhibitors, including tacrolimus, are thoughtto suppress proinflammatory cytokine production andexpression of adhesive molecules of neutrophils and PMNcell infiltration (18). In the present study, histopathologicevaluation revealed that aortic I/R injury caused significantdamages in rat uterine tissue as demonstrated by the conges-tion of vessels, disruption of cells in the glandular epithelium,and severe accumulations of PMN cells in the lamina propria.In contrast, a reduced population of PMN cells, regeneratedmorphology of lamina propria, and glandular structures inboth of the tacrolimus-treated groups were observed, espe-cially more pronounced in the pre-ischemia group. Consistent

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FIGURE 3

Analysis of semithin sections of rat uterine tissue. (A) Control group: regular contour of glandular epithelium (arrowheads), lamina propria (*), andfibroblasts (f). (B) Ischemia-reperfusion group: severe edema in lamina propria (*), cytoplasmic edema and deterioration in glandular cells(arrowheads) with some pyknosis (arrows), fibroblastic degeneration (f) in lamina propria. (C) Pre-ischemia tacrolimus group: slight alteration inglandular cellular morphology (arrowheads), a pyknotic cell (arrow), mild disruption of lamina propria (*), fibroblast (f). (D) Post-ischemiatacrolimus group: reduced disruption in glandular cellular structures (arrowheads) with cytoplasmic edema, pyknotic cell (arrow), decreasededema, and reorganization of lamina propria (*). Staining: toluidine blue.Sahin. The effects of tacrolimus on rat uteri. Fertil Steril 2014.

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with our findings, Garcia-Criado et al reported that treatmentwith tacrolimus 4 hours before ischemia attenuated PMNinfiltration and down-regulated free radical formation in livertissue as well (37). In a recent study, Akhi et al demonstratedthat the transplanted uteri of tacrolimus-treated rats had lownumbers of leukocytes, similarly to the control subjects, aswell as reduced numbers of cytotoxic T cells (CD8 positive)confirmed by immunoperoxidase staining. They showedthat tacrolimus effectively suppresses uterine rejection afterallogenic UT in the rat (38).

TABLE 1

Semiquantitative scoring of uterine tissue.

Degenerative featuresGroup

IGroupII

GroupIII

GroupIV

Glandular cell pyknosis þ þþþ þ þþGlandular cell detachment þ þþþþ þþ þþEndometrial epithelium

degenerationþ þþþ þ þþ

Stromal leukocytosis þþ þþþþ þþ þþþNote: þ ¼ present but trivial; þþ ¼ minimal change; þþþ ¼ moderate change; þþþþ ¼severe change.

Sahin. The effects of tacrolimus on rat uteri. Fertil Steril 2014.

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Based on the data accumulated from earlier studies, ta-crolimus attenuates inflammatory response in I/R injury byseveral mechanisms, including inhibition of nuclear factorkB and proinflammatory cytokine mRNA production (39,40). It has been shown that administration of tacrolimusbefore ischemia prevents mitochondrial dysfunction duringhypoxia and thereby precludes decreasing ATP content inischemic cells (22, 41). In the present study, morepronounced protective effects of tacrolimus in thepreischemia group might be related to better preservationof cellular ATP content compared with the post-ischemiagroup.

In this study, we evaluated the effects of tacrolimuson rat uteri exposed to 30 minutes of ischemia and 1hour of reperfusion. Future studies are needed to comparethe impacts of longer warm ischemia and reperfusiontimes on rat uterine tissue. Also, molecular aspects of ta-crolimus protective role on uterus I/R injury should beinvestigated.

In conclusion, the results of the present study demon-strate that tacrolimus has protective effects against uterineI/R injury induced by distal abdominal aortic occlusion, andthese cytoprotective effects may be due to its antioxidantand antiinflammatory properties.

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Acknowledgments: The authors thank Lale Turkgeldi forher help in preparing the manuscript.

REFERENCES1. Br€annstr€om M, Wranning CA, Altchek A. Experimental uterus transplanta-

tion. Hum Reprod Update 2010;16:329–45.2. Brinsden PR. Gestational surrogacy. Hum Reprod Update 2003;9:483–91.3. Fageeh W, Raffa H, Jabbad H, Marzouki A. Transplantation of the human

uterus. Int J Gynaecol Obstet 2004;76:245–51.4. Racho El-Akouri R, Wranning CA, Molne J, Kurlberg G, BrannstromM. Preg-

nancy in transplanted mouse uterus after long-term cold ischaemic preser-vation. Hum Reprod 2003;18:2024–30.

5. Ramirez ER, Ramirez Nessetti DK, Nessetti MB, Khatamee M, Wolfson MR,Shaffer TH, et al. Pregnancy and outcome of uterine allotransplantation andassisted reproduction in sheep. Minim Invasive Gynecol 2011;18:238–45.

6. Wranning CA, Akhi SN, Diaz-Garcia C, Br€annstr€om M. Pregnancy after syn-geneic uterus transplantation and spontaneous mating in the rat. Hum Re-prod 2011;26:553–8.

7. Ozkan O, Erman Akar M, Ozkan O, Erdogan O, Hadimioglu N, Yilmaz M,et al. Preliminary results of the first human uterus transplantation from amultiorgan donor. Fertil Steril 2013;99:470–6.

8. Hansen A. Swedish surgeons report world’s first uterus transplantationsfrom mother to daughter. BMJ 2012;345:e6357.

9. Kisu I, MiharaM, Banno K, Umene K, Araki J, Hara H, et al. Risks for donors inuterus transplantation. Reprod Sci 2013;20:1406–15.

10. Kupiec-Weglinski JW, Busttil RW. Ischemia and reperfusion injury in livertransplantation. Transplant Proc 2005;37:1653–6.

11. Gorsucha WB, Chrysanthoub E, Schwaebleb WJ, Stahla GL. The comple-ment system in ischemia-reperfusion injuries. Immunobiology 2012;217:1026–33.

12. Kim J, Jang HS, Park KM. Reactive oxygen species generated by renalischemia and reperfusion trigger protection against subsequent renalischemia and reperfusion injury in mice. Am J Physiol Renal Physiol 2010;298:F158–66.

13. Ersahin M, Ozsavcı D, Sener A, Ozakpınar OB, Toklu HZ, Akakin D, et al.Obestatin alleviates subarachnoid haemorrhage–induced oxidative injuryin rats via its antiapoptotic and antioxidant effects. Brain Inj 2013;27:1181–9.

14. Dobashi K, Ghosh B, Orak JK, Singh I, Singh AK. Kidney ischemia-reperfusion: modulation of antioxidant defenses. Mol Cell Biochem 2000;205:1–11.

15. Vincenti F, Jensik SC, Filo RS, Miller J, Pirsch J. A long-term comparison oftacrolimus (FK506) and cyclosporine in kidney transplantation: evidencefor improved allograft survival at five years. Transplantation 2002;73:775–82.

16. Jain AB, Kashyap R, Rakela J, Starzl TE, Fung JJ. Primary adult liver transplan-tation under tacrolimus: more than 90 months actual follow-up survival andadverse events. Liver Transpl Surg 1999;5:144–50.

17. Jordan ML, Shapiro R, Gritsch HA, Egidi F, Khanna A, Vivas CA, et al. Long-term results of pancreas transplantation under tacrolimus immunosuppres-sion. Transplantation 1999;67:266–72.

18. St. Peter SD, Moss AA, Mulligan DC. Effects of tacrolimus on ischemia-reperfusion injury. Liver Transpl 2003;9:105–16.

19. Pratschke S, Bilzer M, Gr€utzner U, Angele M, Tufman A, Jauch KW, et al. Ta-crolimus preconditioning of rat liver allografts impacts glutathione homeo-stasis and early reperfusion injury. J Surg Res 2012;176:309–16.

20. Wakabayashi H, Karasawa Y, Tanaka S, Kokudo Y, Maeba T. The effect ofFK506 on warm ischemia and reperfusion injury in the rat liver. Surg Today1994;24:994.

21. Marcondes FK, Bianchi FJ, Tanno AP. Determination of the estrous cyclephases of rats: some helpful considerations. Braz J Biol 2002;62:609–14.

1182

22. Laurens M, Scozzari G, Patrono D, St.-Paul MC, Gugenheim J, Huet PM,et al. Warm ischemia–reperfusion injury is decreased by tacrolimus in stea-totic rat liver. Liver Transpl 2006;12:217–25.

23. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues bythiobarbituric acid reaction. Anal Biochem 1979;95:351–8.

24. Beutler E, Duron O, Kelly BM. Improved method for the determination ofblood glutathione. J Lab Clin Med 1963;61:882–8.

25. Totsuka E, Fung JJ, Hakamada K, Ohashi M, Takahashi K, Nakai M, et al. Syn-ergistic effect of cold and warm ischemia time on postoperative graft func-tion and outcome in human liver transplantation. Transplant Proc 2004;36:1955–8.

26. Díaz-García C, Akhi SN, Martínez-Varea A, Br€annstr€om M. The effect ofwarm ischemia at uterus transplantation in a rat model. Acta Obstet GynecolScand 2013;92:152–9.

27. Wranning CA, El-Akouri RR, Lundmark C, Dahm-K€ahler P, M€olne J,Enskog A, et al. Auto-transplantation of the uterus in the domestic pig(Sus scrofa): Surgical technique and early reperfusion events. J Obstet Gy-naecol Res 2006;32:358–67.

28. Wranning CA, Dahm-K€ahler P, M€olne J, Nilsson UA, Enskog A,Br€annstr€om M. Transplantation of the uterus in the sheep: oxidative stressand reperfusion injury after short-time cold storage. Fertil Steril 2008;90:817–26.

29. Wranning CA,M€olne J, El-Akouri RR, Kurlberg G, Br€annstr€omM. Short-termischaemic storage of human uterine myometrium—basic studies towarduterine transplantation. Hum Reprod 2005;20:2736–44.

30. Suzuki S, Toledo-Pereyra LH, Rodriguez FJ, Cejalvo D. Neutrophil infiltrationas an important factor in liver ischemia and reperfusion injury. Modulatingeffects of FK506 and cyclosporine. Transplantation 1993;55:1265–72.

31. Perrea DN, Moulakakis KG, Poulakou MV, Vlachos IS, Papachristodoulou A,Kostakis AI. Correlation between oxidative stress and immunosuppressivetherapy in renal transplant recipients with an uneventful postoperativecourse and stable renal function. Int Urol Nephrol 2006;38:343–8.

32. Ross D. Glutathione, free radicals and chemotherapeutic agents. Mecha-nisms of free-radical induced toxicity and glutathione-dependent protec-tion. Pharmacol Ther 1988;37:231–49.

33. Glantzounis GK, Salacinski HJ, Yang W, Davidson BR, Seifalian AM. Thecontemporary role of antioxidant therapy in attenuating liver ischemia-reperfusion injury: a review. Liver Transpl 2005;11:1031–47.

34. Nishinaka Y, Sugiyama S, Yokota M, Saito H, Ozawa T. Protective effect ofFK506 on ischemia/reperfusion–induced myocardial damage in canineheart. J Cardiovasc Pharmacol 1993;21:448–54.

35. Yabe Y, Koyama Y, Nishikawa M, Takakura Y, Hashida M. Hepatocyte-spe-cific distribution of catalase and its inhibitory effect on hepatic ischemia/re-perfusion injury in mice. Free Radic Res 1999;30:265–74.

36. Chen B, Tang L. Protective effects of catalase on retinal ischemia/reperfusioninjury in rats. Exp Eye Res 2011;93:599–606.

37. Garcia-Criado FJ, Lozano-Sanchez F, Fernandez-Regalado J, Valdunciel-Garcia JJ, Parreno-Manchado F, Silva-Benito I, et al. Possible tacrolimus ac-tion mechanisms in its protector effects on ischemia-reperfusion injury.Transplantation 1998;66:942–3.

38. Akhi SN, Diaz-Garcia C, El-Akouri RR, Wranning CA, M€olne J,Br€annstr€om M. Uterine rejection after allogeneic uterus transplantation inthe rat is effectively suppressed by tacrolimus. Fertil Steril 2013;99:862–70.

39. Krishnadasan B, Naidu B, Rosengart M, Farr AL, Barnes A, Verrier ED, et al.Decreased lung ischemia-reperfusion injury in rats after preoperative admin-istration of cyclosporine and tacrolimus. J Thorac Cardiovasc Surg 2002;123:756–67.

40. Squadrito F, Altavilla D, Squadrito G, Saitta A, Deodato B, Arlotta M, et al.Tacrolimus limits polymorphonuclear leucocyte accumulation and protectsagainst myocardial ischaemia-reperfusion injury. J Mol Cell Cardiol 2000;32:429–40.

41. Kaibori M, Inoue T, Tu W, Oda M, Kwon AH, Kamiyama Y, et al. FK506, butnot cyclosporin A, prevents mitochondrial dysfunction during hypoxia in rathepatocytes. Life Sci 2001;69:17–26.

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