J Huazhong Univ Sci Technol［Med Sci］ DOI 10.1007/s11596-014-1337-6 34(5):687-691,2014 J Huazhong Univ Sci Technol［Med Sci］ 34(5):2014

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Pathological Characteristics of Liver Allografts from Donation after Brain Death Followed by Cardiac Death in Pigs* Hui YE (叶 晖)1†, Dong-ping WANG (王东平)1†, Chuan-zhao ZHANG (张传钊)1, Long-juan ZHANG (张龙娟)2, Hao-chen WANG (王皓晨)1, Zhuo-hui LI (李焯辉)1, Zhen CHEN (陈 祯)3, Tao ZHANG (张 涛)3, Chang-jie CAI (蔡常洁)4, Wei-qiang JU (鞠卫强)1, Yi MA (马 毅)1, Zhi-yong GUO (郭志勇)1#, Xiao-shun HE (何晓顺)1# 1 Organ Transplant Center, 2Laboratory of Surgery, 3Department of Anesthesiology, 4Surgical Intensive Care Unit, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2014

Summary: Donation after brain death followed by circulatory death (DBCD) is a unique practice in China. The aim of this study was to define the pathologic characteristics of DBCD liver allografts in a porcine model. Fifteen male pigs (25–30 kg) were allocated randomly into donation after brain death (DBD), donation after circulatory death (DCD) and DBCD groups. Brain death was induced by augmenting intracranial pressure. Circulatory death was induced by withdrawal of life support in DBCD group and by venous injection of 40 mL 10% potassium chloride in DCD group. The donor livers were perfused in situ and kept in cold storage for 4 h. Liver tissue and common bile duct samples were collected for hematoxylin and eosin staining, TUNEL testing and electron microscopic examination. Spot necrosis was found in hepatic parenchyma of DBD and DBCD groups, while a large area of necrosis was shown in DCD group. The apoptosis rate of hepatocytes in DBD [(0.56±0.30)%] and DBCD [(0.50 ± 0.11)%] groups was much lower than that in DCD group [(3.78±0.33)%] (P0.05)). The structures of bile duct were intact in both DBD and DBCD groups, while the biliary epithelium was totally damaged in DCD group. Under electron microscope, the DBD hepatocytes were characterized by intact cell membrane, well-organized endoplasmic reticulum, mild mitochondria edema and abundant glycogens. Broken cell membrane, mild inflammatory cell infiltration and sinusoidal epithelium edema, as well as reduced glycogen volume, were found in the DBCD hepatocytes. The DCD hepatocytes had more profound cell organelle injury and much less glycogen storage. In conclusion, the preservation injury of DBCD liver allografts is much less severe than that of un-controlled DCD, but more severe than that of DBD liver allografts under electron microscope, which might reflect post-transplant liver function to some extent. Key words: organ donation; brain death; cardiac death; liver allografts; pathology  Organ transplantation is the only treatment of choice for patients with end-stage organ failure. As techniques standardized and indications expanded, the number of patients on the waiting list for organ transplantation is continuing to increase. On the other side, the number of available donated organs is not increasing correspondingly, leading to organ shortage all over the world. Therefore, the transplant society tries its best to

Hui YE, E-mail: [email protected]; Dong-ping WANG, E-mail: [email protected] † These authors contributed equally to this work. # Corresponding author, Zhi-yong GUO, E-mail: rockyucsf1981 @126.com; Xiao-shun HE, E-mail: [email protected] * This study was supported by grants from the National High Technology Research and Development Program of China (863 Program) (No. 2012AA021008), the Special Fund for Science Research by Ministry of Health (No. 201302009), the Key Clinical Specialty Construction Project of National Health and Family Planning Commission of the People’s Republic of China, the Science and Technology Planning Key Clinical Project of Guangdong Province (No. 2011A030400005), and the Key Laboratory Construction Project of Guangdong Province.

expand the donor pool with the aim to minimize the deleterious effects of organ shortage on patient outcomes. In China, organ shortage is much more serious. As a response, in March 2011, the Chinese Ministry of Health initiated a pilot program for organ donation from deceased citizens[1]. Considering the culture difference, the Chinese authority has designed three categories of deceased organ donation, namely China Categories Ⅰ(Organ Donation after Brain Death, DBD), Ⅱ (Organ Donation after Circulatory Death, DCD) and Ⅲ (Organ Donation after Brain Death followed by Circulatory Death, DBCD)[2]. DBCD is a unique practice in China. In this case, the donor satisfies DBD criteria, but the family members do not accept donation with the heart still beating. As a result, donation is postponed until cardiac function has ceased after withdrawal of life support. Because the concept of brain death has not been widely accepted by Chinese people, DBCD constitutes a large part of organ donation in China. Our transplant center hosts one of the largest deceased organ donation programs in China. Our initial experience shows that with careful donor selection, it is safe to use these organs. However, the incidence of early allograft dysfunction (EAD) is much higher in liver

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transplant recipients than in our historical controls and recipients receiving DBD organs, suggesting distinct pathogenesis among different donation categories. To further investigate the characteristics of DBCD donors, we’ve successfully established a DBCD porcine model. In this study, we aimed to document the preservation injury pattern of the liver allografts from DBCD donors in a pathological aspect, by comparing with those of DBD and DCD liver allografts. 1 MATERIALS AND METHODS 1.1 Animals Fifteen pigs (4–6 months old, 24.8±4.6 kg) were obtained from the Experimental Animal Center of South Medical University, China. The animals were housed at 20°C–25°C and fed with a standard food. The animals were free to drinking water. All animals received humane care according to the National Guidelines for the Care of Animals in China. The experimental protocol was approved by the local committee for animal experiments. 1.2 Anesthesia All pigs were fasted for 12 h and had only access to water during the night before experiments. These animals received a premedication with intramuscular injection of ketamine (10 mg/kg) in combination with fentanyl (4 μg/kg). After being sedated, the animals were secured in supine position and intubated via a tracheostomy under anesthesia with 5% isoflurane and atracurium (0.5 mg/kg, injected through the venous access). During surgery, the concentration of isoflurane was maintained at 2% and volume-controlled mechanical ventilation (tidal volume, 8 mL/kg; oxygen flow, 1–2 L/min; respiratory rate, 20 beats per min) was applied. 1.3 Grouping 1.3.1 DBD Group The pigs in DBD group (n=5) experienced continuous high intracranial pressure formed by an expanding balloon to induce brain death. After 12 h of observation, organ procurement was performed following the second confirmation of brain death. 1.3.2 DBCD Group Brain death of the pigs in DBCD group (n=5) was induced by using the same way as in DBD group. After the brain death was confirmed again after a 12-h period, cardiac death was induced by withdrawing ventilatory support. A 5-min “no touch” time was used before organ harvesting. 1.3.3 DCD Group The pigs in DCD group (n=5) used venous injection of 40 mL 10% potassium chloride to induce cardiac arrest and organ procurement was performed at 1 h after cardiac death. 1.4 Organ Procurement and Preservation The donor livers were perfused in situ with 1 L hypertonic citrate adenine solution and 1 L Celsior solution at 4°C after cannulation and ligation of the abdominal aorta and portal vein. Isolated organs were stored in an ice box with Celsior solution at 4°C for 4 h. All tissue samples were collected from the same lobe of the livers. And the biliary biopsies were taken from the common bile ducts at 1–2 cm distal to the endings. 1.5 Light Microscopy All biopsies were fixed in 10% formalin, embedded in paraffin, and sectioned at 5 μm. The sections were de-

J Huazhong Univ Sci Technol［Med Sci］ 34(5):2014

paraffinized and dehydrated, and then stained with hematoxylin and eosin (H&E). The extent of hepatocyte vacuolization, necrosis, sinusoidal congestion and biliary integrity was examined. Histological changes of different liver biopsies were scored from 0 to 4 (Suzuki score) according to the severity of hepatocyte vacuolization, parenchymal cellular necrosis and sinusoidal congestion[3]. 1.6 Electron Microscopy Liver biopsy samples of 1 mm3 in size were fixed in 2.5% glutaraldehyde containing 0.1 mol/L phosphate and 2% paraformaldehyde, and then postfixed in 1% osmium tetroxide with sodium cacodylate buffer for 1.5 h. After washed in phosphate buffer, fragments were dehydrated in graded alcohols and embedded in acetone. Thin sections of 60–80 nm-thick were cut from the larger section using an ultramicrotome, and stained with uranyl acetate and lead citrate. All samples were examined and photographed with transmission electron microscope (FEI Tecnai G20, USA). 1.7 Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-End Labeling (TUNEL) Assay TUNEL assay was used to analyze apoptosis of hepatic parenchymal cells with an in situ Cell Death Detection kit (Roche Applied Science, USA) following the manufacturer’s instruction. Quantitative analysis was presented as number of TUNEL-positive hepatocyte nuclei per five hundred hepatocyte nuclei in each group. 1.8 Statistical Analysis The data were analyzed using SPSS 13.0 statistical package (Chicago, Illinois, USA). All values were expressed as ±s. One-way ANOVA analysis was used to determine the significance of differences. A P value less than 0.05 was considered as statistical significance. 2 RESULTS 2.1 Hepatocyte Injury After 4 h of static cold storage, different histological changes were shown in the three groups. Livers from DBD and DBCD groups demonstrated well-organized hepatocyte cords, while in DCD group the continuity of partial hepatocyte cords was broken. Mild cellular edema of hepatic parenchymal cells and sinusoidal space swelling were observed in DBD and DBCD groups and hepatocyte vacuolization was rare. In DCD groups, cellular edema and sinusoidal space swelling were more severe and the extent of hepatocyte vacuolization was larger than in DBD and DBCD groups. Compared with DBD and DCD groups, more severe sinusoidal congestion was observed in the DCD group (fig. 1A). Only spot necrosis in hepatic parenchyma was shown in DBD and DBCD groups, while a large area of necrosis with infiltration of inflammatory cells was observed in DCD group. The necrotic extent of hepatocytes in DBD and DBCD groups was significantly less than that in DCD group (fig. 1B). Taken together, the severity of liver injury in DBD (Suzuki sores: 2.40±0.89) and DBCD (3.00±0.71) groups was significantly less than that in DCD group (5.20±0.48) (P0.05) (fig. 1C).

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Fig. 1 Pathologic characteristics of hepatic parenchyma in DBD, DBCD and DCD groups A: Necrotic extent of hepatocytes in DBD, DBCD and DCD groups. The necrotic extent of hepatocytes in DBD and DBCD group was significantly less than in DCD group. B: spot necrosis in DBD and DBCD groups (indicated by black arrows). A large necrotic area in DCD group (indicated by black arrow) (H&E, ×20). C: Suzuki scores (*P