Effect of Polyethylene Glycol in Pig Intestinal Allotransplantation Without Immunosuppression

Journal of Surgical Research 176, 621–628 (2012) doi:10.1016/j.jss.2011.10.012

Effect of Polyethylene Glycol in Pig Intestinal Allotransplantation Without Immunosuppression Thierry Yandza, M.D., Ph.D.,*,†,‡,1 Michel Tauc, Ph.D.,§ Danielle Canioni, M.D.,{ Claire Rogel-Gaillard, Ph.D.,k,#,** Ghislaine Bernard, M.D., Ph.D.,‡,†† Alain Bernard, M.D., Ph.D.,‡,†† and Jean Gugenheim, M.D., Ph.D.† *P^ ole Digestif, Unite de Support Nutritionnel et de Greffes Intestinales, 3eme B, H^opital de, l’Archet 2, Centre Hospitalo-Universitaire de Nice, Nice, France; †P^ole Digestif, Service de Chirurgie Digestive et Centre de Transplantation Hepatique, H^opital de l’Archet 2, Centre Hospitalo-Universitaire de Nice, Nice, France; ‡INSERM U576, Faculte de Medecine, Nice, France; §Laboratoire CNRS 3093, Universite de Nice-Sophia Antipolis, Nice, France; {Service d’anatomopathologie, H^opital Necker-Enfants malades, Universite Paris V, France; kINRA, UMR 1313 de Genetique Animale et Biologie Integrative, Domaine de Vilvert, Jouy-en-Josas, France; #AgroParisTech, UMR 1313 de Genetique Animale et Biologie Integrative, Domaine de Vilvert, Jouy-en-Josas, France; **CEA, DSV, IRCM, SREIT, Laboratoire de Radiobiologie et Etude du Genome, Domaine de Vilvert, Jouy-en-Josas, France; and ††Laboratoire d’Immunologie, H^opital de l’Archet 2, Centre Hospitalo-Universitaire de Nice, Nice, France Originally submitted March 24, 2011; accepted for publication October 13, 2011

Objectives. We evaluated whether IGL-1, a graft preservation solution containing polyethylene glycol, improves the outcome of small bowel grafts in comparison to the University of Wisconsin (UW) solution in a pig allotransplantation model. Materials and Methods. Seventeen pigs were randomly allocated to group 1 (n [ 10; intestinal allotransplantation with IGL-1) and group 2 (n [ 7; allotransplantation with UW). Pigs received no immunosuppression and were sacrificed on postoperative d (POD) 8. Intestinal specimens were obtained from the animal immediately before cold flushing (T0), 2 h after graft reperfusion (T1), and at sacrifice (T2). Results. Survival rate to POD 8 was 50% in group 1 compared with 16% in group 2 (P < 0.05); 62% of pigs in group 1 did not present any acute cellular rejection (ACR) compared to 16% in group 2 (P < 0.05). Severe ACR rate was 25% in group 1 and 66% in group 2 (P < 0.05). iNOS activity and intestinal caspase 3 levels increased significantly between T0 and T1 in group 1 compared to group 2 (P < 0.05). Cell necrosis increased significantly between TO and T1 in group 2 compared with group 1 (P < 0.05) whereas cell apoptosis was significantly higher at T1 compared with T0 in group 1 in comparison to group 2.

1 To whom correspondence and reprint requests should be addressed at Service de Chirurgie Digestive et Centre de Transplantation H epatique, H^ opital l’Archet 2, 151 route de Saint Antoine de Ginestiere 06202 Nice Cedex 3 France. E-mail: [email protected].

Conclusions. Our results show that IGL-1 improves intestinal graft viability as compared to UW solution, possibly by reducing graft immunogenicity and by favoring intestinal epithelial repair. Ó 2012 Elsevier Inc. All rights reserved.

Key Words: acute cellular rejection; ischemia-reperfusion-injury; intestinal transplantation; pig; polyethylene glycol; IGL-1; UW.

INTRODUCTION

Intestinal transplantation has become the therapy of choice for patients with irreversible intestinal failure who have developed life-threatening complications or have otherwise failed with total parenteral nutrition [1]. Despite improved early patient survival rate at 1 year now exceeding 90% at experienced centers, the 5y survival rate remains low, around 50%. Major obstacles to the development of reliable and safe small bowel (SB) transplantation are primarily those of bacterial infection and allograft acute cellular rejection (ACR) [2]. Both clinical and experimental data demonstrate that events occurring at the time of transplantation, called ischemia reperfusion injury (IRI), may have deleterious short and long term effects, manifesting as increased episodes of acute rejection and chronic allograft dysfunction [3]. Recently, the acute phase of IRI has been increasingly viewed as part of the innate immune response to the lack of vascular perfusion and oxygen

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establishing a link between innate immunity, adaptive immune responses and organ regeneration, and thus effecting long-term graft function [4]. For these reasons, we postulate that the composition of preservation solutions is critical not only for the preservation and the quality of organs used for transplantation, but also for the immunogenicity of the graft. To date, preservation protocols are predominantly directed by the fact that SB is harvested as part of a multiorgan procurement procedure that utilizes a common vascular preservation solution. Currently, the University of Wisconsin solution (UW) remains the gold standard vascular perfusate [5]. However, UW has not proven its superiority in comparison to other preservation solutions in SB transplantation as equivalent results have been achieved with simple crystalloid solutions such as normal saline and lactated Ringer’s solution [6]. Furthermore, severe histologic alterations of graft mucosa have been noted after short periods of preservation by UW in humans [7]. Finally, some of the properties of UW solution do not favor organ preservation. For example, previous studies have shown that the performance of UW solution is limited by its adjunction of hydroxyethyl starch (HES) that has hyperaggregating effect on rat and human red blood cells [8]. Its high potassium concentration could damage cells and induce vasoconstriction [9]. Thus, it is rather by default that the current clinical standard for SB consists of a vascular flush with UW solution as part of multivisceral organ procurement. Recently, a modified UW preservation solution called Institut Georges Lopez (IGL-1), characterized by the inversion of Kþ and Naþ concentrations and the replacement of hydroxyethyl starch by polyethylene glycol (PEG20) in the original UW solution, has been successfully used in pig and human kidney transplantation [10] and for human liver preservation [11]. PEG is a nontoxic, flexible, water-soluble polymer that is supposed to combine two protective actions: an oncotic pressure that limits the deleterious effects of edema [12] and stabilization of membrane lipids that enhance the immunoprotection of donor cells, tissues, and organs [13]. The first published work concerning the immunosuppressive effect of PEG20 containing solution in man is from the Collins et al. group in 1991 [14]. They reported that introduction of PEG20 (50 g/L) in a modified St. Thomas solution (Cardiosol) could significantly lower the rate of acute rejection in heart transplantation. Other groups have observed the same effect in experimental surgery when replacing hydroxyethyl starch by PEG20 in a modified UW solution in liver [15], pancreas [16], and kidney transplantation. The purpose of the present study was to evaluate the use of IGL-1 on intestinal transplantation in pigs

without immunosuppression, compared with standard UW solution. Moreover, we aimed to understand its mechanisms of action in this setting. METHODS Study Groups The study protocol was approved by The Institutional Committee for Animal Research of the University of Nice. The ‘‘Principles of Laboratory animal care’’ [17, 18] were followed. Thirty-four female outbred (one donor, one recipient), non-related Large White_X_ Landrace pigs were used. Recipients were randomly allocated to two study groups. Pigs in group 1 (IGL-1 group, n ¼ 10), received an intestinal orthotopic allotransplantation with IGL-1 as the preservation solution. Group 2 (n ¼ 7) consisted of recipients receiving an intestinal orthotopic allotransplantation with UW as the preservation solution. No immunosuppression was used. Animals were fasted for 24 h before surgery but they were allowed free access to water. In both groups, the surviving animals were sacrificed on postoperative d 8 (POD8).

Anesthesia The animals were anesthetized using the following protocol: intramuscular premedication with 20–25 mg/kg ketamine (Imalgene 1000, 10 mL; Merial, Lyon, France), and 0.01–0.02 mg/kg glycopyrrolate (Robinul V, 5 mL; Vetoquinol, Lure, France); induction with isoflurane gas (AErrane; Baxter SA, Maurepas, France) administered with a mask. The animals were intubated using a laryngoscope and endotracheal tube (Hudson RCI, Sheridan/CF, ID 6.0–7.0 mm) and mechanically ventilated (Veterinary Anesthesia Ventilator model 2000; Hallowell FC) (respiratory rate: 15–20/min; FIO2 ¼ 30%). Anesthesia was maintained with repeated doses of ketamine and fentanil (Fentanyl Dakota Pharm, 0.1 mg/2 mL; Le Plessis Robinson, France) intravenously. During the operation, 50 mL/kg saline (NaCl 0.9%; B. Braun, Boulogne, France) and 65 mL/kg glucose 5% (Glucose Isotonique Aguettant; Aguettant, Lyon, France) were infused intravenously. Before the abdominal incision, a venous catheter was inserted into the external jugular vein for central venous pressure monitoring and perfusion. The arterial blood pressure was monitored (SC 7000; Siemens, Danvers, MA) through a cervical catheter inserted into the carotid artery. Heart rate and O2 saturation percentage were monitored continuously using a pulse oxymeter with the sensor attached to the gum (model 9847 V; Nonin Medical Inc., Plymouth, UK).

Allotransplantation The technique of allotransplantation has been described elsewhere [18]. Briefly, in the donor, the isolated jejunoileum was flushed with 1000 mL of cold (4 C) preservation solution through the infrarenal aorta. The graft consisted of the entire jejunum and ileum, the superior mesenteric artery (SMA) in continuity with the abdominal aorta, the superior mesenteric vein (SMV) in continuity with the portal vein. The graft was kept in the chosen cold preservation solution bath before transplantation. The small bowel lumen was not flushed. In the recipient, the jejunoileum was removed. The allograft was implanted by anastomosing the donor abdominal aorta to the recipient infrarenal aorta and the donor SMV to the recipient infra-renal inferior vena cava. Intestinal continuity was restored using end-to-end anastomoses of the jejunal and ileal transections.

Postoperative Care At the end of surgery, a single intramuscular dose of 4 mg/kg of tolfenamic acid (Tolfedine 4%; Vetoquinol, Lure, France) was administered to animals for the postoperative analgesia and a single

YANDZA ET AL.: PEG20 IN A PIG INTESTINAL TRANSPLANTATION MODEL intramuscular dose (15 mg/kg) of long-acting amoxicillin (Clamoxyl L.A; Pfizer) was given as an antimicrobial prophylaxis. Animals were extubated after the operation and placed in metabolic cages with heat lamps. Every animal received intravenously 250 mL glucose 5% on the first and the second day following surgery using the central venous catheter left in the external jugular vein. Fluids and commercial foods (Mini-porcs 127; Maintenance Diets Safe, Augy, France) were allowed orally on the first postoperative day. Animals were euthanized on the postoperative d 8 with intravenous overdose of sodium pentobarbital (Dolethal; Vetoquinol SA, Lure, France) after a controlled laparotomy under gas anesthesia using isoflurane.

Monitoring The survival time, stool appearance and appearance of the intestine at relaparotomy at postoperative d 8 were monitored. Specimens for histologic, immunologic, and immunohistochemical studies were obtained from the animal immediately before cold flushing (T0 or control), 2 h after graft reperfusion (T1), and at POD8 (T2).

Composition of Solutions The composition of the preservation solutions is shown in Table 1. UW solution (Viaspan; Bristol-Myers Squibb, Brussels, Belgium) is the original Belzer solution without dexamethasone, insulin, or antibiotics [5]. IGL-1 solution is a modified UW solution manufactured by the Institut Georges Lopez in Lyon, France, which was obtained by replacing hydroxyethyl starch with PEG 20 and changing the concentrations of Naþ and Kþ.

Histologic Study Intestinal specimens were fixed with 10% buffered formalin, embedded in paraffin, cut into 5-mm sections, and stained with hematoxylin and eosin (HE). All specimens were analyzed by light microscopy by a single pathologist to whom the status of the specimen was unknown. The degree of histologic injury in the small bowel was assessed using the Park et al. scale [19]: 0, normal histology; 1, slight disruption of the surface epithelium; 2, epithelial cell loss and injury at villus tip; 3, mucosal vasocongestion, hemorrhage, and focal necrosis with loss of less than one-half of villi; and 4, damage extending to more than one-half of villi. The degree of apoptosis was assessed according to the number of apoptotic bodies observed in the intestinal crypts after reperfusion and was scored as: 1 (less than three apoptotic bodies per crypt), 2 (three

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to five apoptotic bodies per crypt), and 3 (more than five apoptotic bodies per crypt). Grading of acute cellular rejection was the following: (a) grade 0: specimen characteristics are indistinguishable from normal bowel histology; (b) grade 1 or indeterminate for rejection: minimal lesions consisting of <6 apoptotic cells out of 10 crypts and few or no inflammatory lesions; (c) grade 2 or mild rejection: >6 apoptotic cells out of 10 crypts associated with a mild to moderate inflammatory infiltrate of predominantly mononuclear cells; (d) grade 3 or moderate rejection: increased apoptotic cells which become confluent and may lead to crypt destruction; associated with moderate to severe inflammatory infiltrate of mononuclear cells; (e) grade 4 or severe rejection: more important crypt destruction with areas with vanishing crypts or mucosal erosions or ulcerations; severe inflammatory infiltrate [20].

Caspase 3 and Nitric Oxide Synthase Activities The mucosa of the intestinal segment (10–15 cm) was scraped from the underlying muscular layers on ice in cold free Ca2þ and Mg2þ PBS. After centrifugation (350 3 g, 3 min) the pellet was homogenized with a Kinematica Polytron homogenizer at 4 C in the following buffer (2 mL/g wet weight): (in mM) mannitol 300, EGTA 5, Tris 12 pH 7.4. Enzymatic activities were determined directly on this fresh homogenate. Caspase 3 activity was determined spectrophotometrically at 405 nm by measuring cleavage of the specific caspase 3 substrate N-acetylAsp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (Ac-DEVDAFC) in PNA using the Sigma Caspase 3 kit (Caspase 3 Activity Assay; Sigma-Aldrich, St. Louis, MO). Nitric oxide synthase (NOS) activity was assayed spectrophotometrically (Colorimetric NOS assay kit; Oxford Biochemical Research, Oxford, MI) by measuring the accumulation of its stable degradation product nitrite in the presence of nitrate reductase. Total proteins were determined at 595 nm with the classic Bio-Rad assay kit II.

Swine Leucocyte Antigen (SLA) Typing Histocompatibility status of the donors and recipients was assessed by allele characterization of the three classical SLA class I loci (SLA-1, SLA-2, and SLA-3) using the PCR sequence-specific primer strategy described by Ho et al. [21]. DNA was extracted from peripheral blood mononuclear cells and lymph node cells for recipients and donors, respectively. Following allele group nomenclature, animals harboring alleles belonging to distinct groups for at least one gene were considered as noncompatible.

Statistical Analysis TABLE 1 Composition of UW and IGL-1 Preservation Solutions

HES (mmol/L PEG-35 (mmol/L) Lactobionic acid (mmol/L) Raffinose (mmol/L) MgSO4 (mmol/L) KH2PO4 (mmol/L) Glutathione (mmol/L) Adenosine (mmol/L) Allopurinol (mmol/L) Naþ (mmoml/L) Kþ (mmol/L) Osmolality (mOsm/Kg) pH

UW

IGL-1

0.25 – 100 30 5 25 3 5 1 30 125 320 7.2–7.4

– 0.03 100 30 5 25 3 5 1 125 30 320 7.2–7.4

Values were expressed as mean 6 standard error of the mean (SEM). The Mann and Whitney test was used for comparisons between groups and the Wilcoxon Rank test was used for comparisons between T0, T1, and T2. Comparison of survival rate between groups was made by Fischer’s exact test. A P value corresponding to a P < 0.05 was considered to be significant.

RESULTS

Three animals that died before the third postoperative day (two in group 1 and one in group 2) were excluded from the study. The causes of death were perioperative bleeding in one case and vascular thrombosis in two cases. Thus, results of eight pigs in group 1 and six pigs in group 2 were included in the study.

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Peroperative Parameters

The mean weight at the time of transplantation, the mean duration of the operation, and the mean cold ischemia time did not significantly differ between both groups (Table 2). Postoperative Clinical Outcome

All surviving animals started spontaneous feeding the first day after surgery. In both groups, the first stool occurred between the second and the third day after surgery. In group 1, four animals survived until the postoperative d 8 and were sacrificed, according to the protocol (Table 3). Intestinal biopsy performed on the ileum did not show ACR in these four animals. Four pigs died before POD8. At laparotomy, three pigs had intestinal perforation and peritonitis and the remaining one had pulmonary embolism. In all four animals, biopsy of the ileum showed an ACR. In group 2, one animal survived until POD8 with a normal ileum histology on POD8. Five pigs died before POD8 from intestinal perforation and peritonitis. Histology of the ileum done at autopsy showed ACR in all five pigs. Thus, the survival rate on POD8 was 50% in group 1 compared with 16% in group 2 (P < 0.05). SLA Typing

SLA typing showed SLA mismatches in all cases but one where typing was detected as likely semicompatible (one pig in group 1, which had no intestinal rejection and lived until sacrifice on POD8). Histologic Findings (Figs. 1 and 2)

Two hours after reperfusion (T1), histologic lesions observed in the intestinal mucosa in the intestinal crypts differed between both groups (Table 4). Tissue damage according to Park’s classification was worst in group 2 compared with group 1. Epithelial necrosis was seen in both experimental groups, with the worst damage found in group 2 (3.50 6 0.54 versus 2.33 6 0.82, P < 0.05) (Table 4). The injury in group 2 was extensive (more than one-half of

the villi for select samples), and included hemorrhage and mucosal vasocongestion. Regarding the degree of apoptosis in the intestinal crypts after reperfusion, the highest number of apoptotic bodies was found in group 1 (Table 4). All pigs had intestinal biopsies of the ileum at death. The earliest lesions of ACR were observed on POD4. In group 1, histologic studies on POD8 showed the absence of ACR in five pigs (62.5%), and an ACR in three animals (37.5%), including one moderate ACR (12.5%) and two severe ACR (25%) (Table 5). In group 2, one pig (16.6%) had no ACR, whereas five animals (83.4%) experienced an ACR consisting of one moderate ACR (16.7%) and four severe ACR (66.7%) (P < 0.05). There was a correlation between the macroscopic aspect at laparotomy and the histologic findings. In the absence of acute cellular rejection, there were no adhesions. The small bowel appeared normal. The mesenteric lymph nodes were not enlarged. In contrast, in the case of acute cellular rejection, the transplanted bowel formed a hard, whitish, and irregular mass with dense adhesions. Mesenteric lymph nodes were massively hypertrophic. On cut section, mesenteric lymph nodes were hemorrhagic and thrombotic. Enzymatic Activities

Caspase-3-like activity was used as an earlier indicator of apoptosis. Intestinal caspase 3 levels increased significantly between T0 and T1 in group 1 (from 41.5 6 6.9 nmol/min mg prot at T0 to 49.9 6 8.9 nmol/ min mg prot at T1; P < 0.05) compared with group 2 (from 31.1 6 9.9 nmol/min mg prot at T0 to 32.9 6 7.0 nmol/min mg prot at T1; NS) (Fig. 3). iNOS activity increased significantly from 0.805 6 0.028 nmol/h/mg prot at T0 to 0.957 6 0.136 nmol/h/mg at T1 in group 1 (P < 0.03), whereas it decreased significantly from 0.793 6 0.116 nmol/h/mg at T0 to 0.661 6 0.096 nmol/ h/mg at T1 in group 2 (P < 0.03) (Fig. 4). DISCUSSION

Our results suggest that IGL-1 strongly reduces intestinal rejection and improves graft viability compared

TABLE 2 Perioperative Parameters in Both Groups Parameters

Group 1 (n ¼ 8)

Group 2 (n ¼ 6)

P

Mean weight (kg 6 SEM*) Mean duration of the operation in the recipient (min 6 SEM) Mean cold ischemia time (min 6 SEM)

25.8 6 7.6 (Range: 19–37) 242.0 6 28.6 (Range: 215–300)

20.6 6 2.7 (Range: 18–26) 252.1 6 41.8 (Range: 185–305)

NS NS

239.7 6 18.8 (Range: 210–260)

243.8 6 16.8 (Range: 218–270)

NS

SEM ¼ standard error of the mean.

*

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TABLE 3 Survival on Postoperative D 8 (POD 8)

Survival on POD 8 Causes of death

Group 1 (n ¼ 8)

Group 2 (n ¼ 6)

P

4/8 (50%) 3 acute cellular rejections (POD 4, 5, 5) 1pulmonary embolism (POD 7)

1/6 (16%) 5 acute cellular rejections (POD 4, 4, 5, 5, 6)

<0.05

to UW solution. This effect may be mostly due to the concepts applied in composition of IGL-1: low-Kþ concentration to avoid vasoconstriction and the use of a new polymer, PEG20, a high-molecular-weight molecule as a colloid [13]. In our study, the improved outcome of intestinal allografts was associated with increased NOS and caspase 3 activities in comparison with UW solution. NOS are a family of enzymes that produce nitric oxide (NO) through the conversion of L-arginine to L-citrulline [22]. NOS exist in constitutive (cNOS) and inducible isoforms. The type 1 or neuronal NOS (nNOS), and type 3 or endothelial NOS (eNOS) are constitutive isoforms and produce low levels of NO continuously under physiologic conditions. In contrast, the type 2 inducible isoform (iNOS) is mostly induced in ischemiareperfusion injury [23]. The physiologic roles of cNOS in the intestine include maintaining intestinal blood flow [23], preventing leukocyte-endothelial adhesion [24], and protection against injury [25]. After ischemia reperfusion injury, the expression of mucosal iNOS is rapidly up-regulated whereas the constitutive isoforms of NOS may be inactivated [26]. Once induced, iNOS produces large amounts of NO for prolonged periods of time. The role of iNOS in ischemia reperfusion injury is controversial. For some authors, NO overproduction owing to increased iNOS activity increases mucosal hyperpermeability, causes mucosal injury, and promotes bacterial translocation [27].

For other authors, mucosal iNOS may play an important role in acute epithelial defence and repair [26] by triggering three local events that culminate in restoration of epithelial continuity and normal permeability: (a) villus contraction, which reduces the total and denuded surface area for repair; (b) migration of enterocytes to seal the exposed basement membrane; and (c) closure of leaky epithelial intercellular spaces and tight junctions. Furthermore, NO favors reestablishment of a normal vascular environment following arterial ischemia reperfusion injury [28] by inhibiting platelet aggregation and adherence, leukocyte adherence, vascular smooth muscle cell (VSMC) proliferation, and VSMC migration. Finally, NO or its by-product, peroxynitrite, may promote apoptosis through multiple potential mechanisms including inducing cytochrome c release with downstream activation of caspases [29]. In our study, IGL-1 promoted significantly apoptosis through caspase 3 production, compared with UW. Apoptosis has been shown to be beneficial after intestinal ischemia reperfusion injury. The central component is a proteolytic cascade involving proteases called caspases [30]. Caspases are not only associated with differentiation, proliferation, and immune functions but are also involved in the regulation of cell survival, proliferation, cell migration, differentiation, and inflammation [30]. The caspases that get activated via recruitment to signalling complexes are known as the initiator caspases, as they provide a link between cell signalling and apoptotic execution. The main initiator caspases

FIG. 1. Histologic sections of the pig small bowel corresponding to different ischemic lesions in this study. These slides are representative examples of the types of ischemic lesions scored in Table 4. (A) Normal architecture and length of the villi (Park’s classification 0-1) representative of T0 in both groups. (B) Total lifting of the villi epithelium (Park’s classification 3) at T1 in group 2. H and E; original magnifications, 310 (A); 3100 (B). (Color version of figure is available online.)

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FIG. 2. Histologic sections of the pig small bowel corresponding to different grades of acute cellular rejection observed in this study. (A) Normal intestinal crypts. (B) Moderate acute cellular rejection characterized by two crypts with confluent apoptotic bodies. (C) Severe acute cellular rejection with confluent apoptotic bodies in the crypts, loss of goblet cells, and basophilic cytoplasm. H and E 31000. (Color version of figure is available online.)

are caspase-2, -8, -9, and -10 in mammals. Caspase-3 is the main downstream effector caspase that cleaves the majority of the cellular substrates in apoptotic cells. During the compensatory proliferation healing process of tissues, caspase-3 controls the final shape and size of tissues to regain their original cell numbers. This may explain the protective action of IGL-1 solution. We found that IGL-1 reduced significantly the necrotic cell death compared to UW. Mitochondria appear to play the key role in the occurrence of either necrosis or apoptosis [31]. The mitochondrial permeability transition (MPT) is the common factor in ischemia/reperfusion that initiates either apoptotic or necrotic cell killing. The fate of the cell is determined at organ reperfusion by the extent of MPT and the availability of adenine 50 -triphosphate (ATP). If MPT is minimal, the cell may recover. If MPT is moderate (10%–50%), the cell may undergo programmed cell death even though energy production is adequate. If MPT is severe

(50%–90%), the cell will die from necrosis due to inadequate energy production [32, 33]. The availability of ATP after reperfusion switches cell killing from necrotic cell death to apoptosis. Cells killed by acute necrotic death can activate resting dendritic cells (DCs) through Toll-like receptors (TLRs) and trigger acute rejection [34], whereas cells dying by physiologic apoptotic death do not [35]. Dutheil et al. [36] have shown that PEG20 contained within IGL-1, preserves ATP content. Thus, IGL-1 may reduce graft immunogenicity by preventing cell necrosis and by stimulating apoptosis, an action that limits DCs maturation and activation through TLR4. Our study may have several limits. First, our mean cold ischemia time is short, being around 4 h, which is different from the usual 6 h cold ischemia time observed clinically. In fact, current trends recommend decreasing cold ischemia time in order to reduce graft ischemia reperfusion injury and to lower graft

TABLE 4 Histologic Lesions Observed in the Intestinal Mucosa in the Intestinal Crypts 2 h(T1) After Reperfusion Groups

Tissue damage (Park’s classification)

Apoptosis at reperfusion

Necrosis at reperfusion

1 2

1–2 3–4

3 1–2

2.33 6 0.82* 3.50 6 0.54*

TABLE 5 Acute Cellular Rejection in the Ileum

Acute cellular rejection

P < 0.05.

*

Group 1 (n ¼ 8)

Group 2 (n ¼ 6)

P

3/8 (37.5%)

5/6 (83.4%)

<0.05

In group 1, there was one moderate ACR (12.5%), and two severe ACR (25%) whereas, in group 2, ACR consisted of one moderate ACR (16.7%) and four severe ACR (66.7%) (P < 0.05).

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an antioxidant mechanism. IGL-1 may favor epithelial repair and reduce ischemia reperfusion injury through iNOS activity. Since the mean ischemia time was relatively short in both groups, we need to evaluate the efficacy of IGL-1 solution in longer ischemia times as well as in a randomized multicenter study to confirm these results in clinics.

ACKNOWLEDGMENTS The authors thank Silvina Ramella-Virieux (Institut Georges Lopez) for providing the IGL-1 solution; Lazzari Anne and Franc¸oise Crechet (CEA-INRA) for their technical assistance; Drs. Janet Maryasnki, Joyce Loeffler, Frederic Berthier, Professors Jean Amiel, Seigo Nishida, Andreas Tzakis, and Jacques Pirenne for their advice and critical review of this manuscript. FIG. 3. Caspase activity. Intestinal caspase 3 levels increased significantly between T0 and T1 in group 1 compared with group 2 (P < 0.05). (Color version of figure is available online.)

immunogenicity [37]. Second, we did not use immunosuppression, in order to determine the PEG20 effect on graft immunogenicity. Thus, one could anticipate whether the effect of IGL-1 may be lost by using calcineurin inhibitors in this model. However, in the study from Collins et al. [14] in a human, PEG20 could lower the acute heart rejection rate despite the use of cyclosporine, a well known calcineurin inhibitor. Thus, we can anticipate that calcineurin inhibitors may not have impacted our study. In conclusion, our results show that IGL-1 improves intestinal graft viability compared with UW solution in intestinal transplantation in pigs. Its mechanism of action involves a reduction of graft immunogenicity by favoring apoptosis instead of cell necrosis and by

FIG. 4. iNOS activity. iNOS activity increased significantly from T0 to T1 in group 1 whereas it decreased significantly from T0 to T1 in group 2 (P < 0.03). (Color version of figure is available online.)

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