Ethical approval for the present study was provided by the Ethical Committee (no. KS1218) of the Shanghai Chest Hospital, affiliated to Shanghai Jiaotong University (Shanghai, China), on 10 July 2011.

The present study was performed in accordance with the Guidelines for Animal Experiments of the Shanghai Jiaotong University (Shanghai, China). Male Sprague-Dawley rats weighing 250–300 g were housed with free access to food and water under a natural day/night cycle. All rats were acclimated for a week prior to any experimental procedures. The rats were anesthetized by an intraperitoneal injection of ketamine chloride (100 mg/kg; Jiangsu Hengrui Medicine Co., Ltd., Lianyungang, China) and intubated through a tracheotomy. Rats were mechanically ventilated with 100% oxygen, at a tidal volume of 10 ml/kg and a respiratory rate of 70 breaths/min. Following heparinization (1000 U/kg intravenously), a median sternotomy was performed. The pulmonary artery (PA) was cannulated through a right ventriculotomy and the left atrium was sheared directly. The lungs were flushed through the PA cannula with 20 ml of cold low-potassium dextran solution (Perfadex, Vitrolife, Gothenburg, Sweden) via gravity drainage of 25 cm H₂O. The PA, vein and trachea were ligated and subsequently the heart-lung bloc was excised from the chest. Following the excision, the inflated lung was placed into low-potassium dextran solution and stored at 4°C for CI preservation. A total of 30 rats were randomly divided into five treatment groups and underwent en bloc heart-lung harvest as described previously (16). These blocs were subsequently followed by CI preservation for varying time periods (0, 3, 6, 12 and 24 h, respectively). Following CI preservation, the left lung tissues of each bloc were fixed in 4% paraformaldehyde (Guangfu Fine Chemical Research Institute, Tianjin, China) in 0.01 M phosphate-buffered saline (PBS; Basic Medicine Faculty of Shanghai Jiaotong University, Shanghai, China) pH 7.4 at 4°C for 24 h. Subsequently, the samples were cryoprotected overnight in 30% sucrose in 0.01 M PBS at 4°C and embedded in optimal cutting temperature (OCT) media prior to immunofluorescence staining. The right lung tissues were frozen in liquid nitrogen and stored at −80°C prior to western blot analysis and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis.

Immunofluorescence staining was performed using transverse sections to investigate alterations in the expression of a specific autophagy marker, microtubule-associated protein 1 light chain 3B (LC3B), at each time point following CI. To identify DNA fragmentation (a marker of apoptosis) in the cells expressing LC3B, double staining of LC3B and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) were performed. Tissue sections (10 μm thickness) were prepared from OCT media-embedded tissue using a cryostat. The sections were washed in 0.01 M PBS and permeabilized in 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). For immunofluorescent labeling, sections were pre-incubated in 20% goat serum (Beyotime Institute of Biotechnology, Nantong, China) for 30 min and subsequently incubated overnight at 4°C with the primary antibody, (LC3B, D11, XP^® rabbit monoclonal antibody used at 1:200; #3868s; Cell Signaling Technology, Inc., Danvers, MA, USA) in 0.01 M PBS. Following three washes in PBS, the sections were incubated for 2 h with Alexa Fluor 488-conjugated anti-rabbit immunoglobulin G antibody (1:500; A11008; Invitrogen Life Technologies, Carlsbad, CA, USA) in 0.01 M PBS at room temperature in the dark. TUNEL staining was applied using an in situ Cell Death Detection kit, TMR red (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer’s instructions. 4′,6-Diamidino-2-phenylindole (Beyotime Institute of Biotechnology) was employed for nuclear staining. Following rinsing in PBS, the sections were mounted with antifade solution (Beyotime Institute of Biotechnology). The immunoreactive signals were sequentially visualized in the same section using an LSM 710 Meta confocal microscope (Zeiss, Jena, Germany) with three distinct filters (405, 488 and 543 nm). The signal intensities through each filter were matched at the time of imaging. The images through each filter were individually optimized for brightness and background prior to generating the final composite images.

The LC3B-positive cells, which were defined as the cells exhibiting punctuate LC3B fluorescent dots (14,17), were counted in the transverse sections scanned previously. A total of six serial sections were used for each sample. The LC3B-positive cells were counted in five random microscopic fields (magnification, ×40) of each section by an examiner who was blinded to the experimental conditions.

Rat lung tissues were homogenized in cold T-PER™ lysis buffer (Pierce Biotechnology, Inc., Rockford, IL, USA) with a protease inhibitor cocktail (Roche Diagnostics GmbH). Subsequently, the lysates were quickly sonicated three times for 15 sec in an ice bath, boiled for 5 min at 95°C and stored at −80°C prior to use. Aliquots of proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel, transferred onto a polyvinylidene difluoride membrane and detected using antibodies against LC3B(D11) XP^® rabbit monoclonal antibody (1:1,000; #3868s; Cell Signaling Technology, Inc.), Beclin-1 (H-300) rabbit polyclonal antibody (1:1,000; sc-11427; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or caspase-3 rabbit polyclonal antibody (1:1,000; #9662s; Cell Signaling Technology, Inc.), respectively. β-actin (1:5,000; Sigma Aldrich, Shanghai, China) was used as a loading control.

RT-qPCR was performed to assess autophagy-related gene 5 (Atg5), B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) gene expression in lung samples. Total RNA was isolated using TRIzol (Invitrogen Life Technologies) according to the manufacturer’s instructions. Reverse transcription was completed using a PrimeScript 1st Strand cDNA Synthesis kit (Takara Biotechnology Co., Dalian, China) according to the manufacturer’s instructions. SYBR^® Green qPCR was performed using an Applied Biosystems 7500 real-time PCR System (Applied Biosystems, Foster City, CA, USA) in a 20 μl reaction containing 2 μl of cDNA, 10 μl of 2X SYBR^® Premix Ex Taq™ (DRR041A, Takara Biotechnology Co.) and primers designed specifically for the genes amplified (18). The primer sequences are shown in Table I (Sangon Biotech, Shanghai, China).

Data are expressed as the mean ± standard deviation. The statistical significance was estimated by one-way analysis of variance followed by the Student-Newman-Keuls test. P<0.05 was considered to indicate a statistically significant difference.

To assess autophagic activity in lung tissues following CI, the protein expression of LC3B-I and LC3B-II (autophagosome marker proteins) was examined using western blot analysis. A significant increase in the ratio of LC3B-II/I was observed after 3 h of CI, peaked at 6 h and subsequently decreased to the basal level after 12 h (Fig. 1A and B). This result suggested that autophagic activity was induced in lung tissues following CI. Consistently, changes in the level of the protein Beclin-1, another key autophagic protein, exhibited a similar trend (Fig. 1C and D).

In order to confirm these findings, the LC3B-positive cells were detected by immunofluorescence staining. A marked increase in the number of LC3B puncta was observed in lung tissue within the prolonged CI time, which is consistent with the trend observed in western blot analysis. The marked increase in LC3B-positive cells was initiated at 3 h, peaked at 6 h following CI and thereafter decreased to the basal level after 12 h of CI preservation (Fig. 2). In order to further confirm autophagy induction in lung tissues following CI, the mRNA level of another important autophagy marker, Atg5, was assessed via qPCR analysis. In a similar manner to that of LC3B induction, a significant increase in Atg5 expression was detected in the 6 h group compared with the 0 h group following CI (Fig. 3).

Autophagic cell death and apoptosis in lung tissues following CI was also determined. Apoptosis in lung tissues following CI was initially verified through analyzing the changes in the expression level of an apoptosis marker enzyme, caspase-3, in each group. The level of cleaved caspase-3, the active form, markedly increased between 6 and 24 h post-CI compared with the basal level at 0 h (Fig. 4A and B). In addition, the ratio of mRNA of the pro-/anti-apoptotic genes Bax/Bcl-2 significantly increased between 6–12 h of CI preservation (Fig. 4C). The present results verified marked apoptosis in the lung CI model. To identify autophagic cell death and apoptosis in lung tissues following CI, double-immunofluorescence analysis of LC3B and TUNEL (an apoptosis-specific staining assay) was performed. The results clearly demonstrated that the levels of TUNEL-positive cells and the LC3B-positive cells increased at 6 h after CI, compared with the 0 h group (Fig. 5). The LC3B-positive signals only partially colocalized with the TUNEL signal when merged. Notably, the nuclei of the TUNEL-positive, LC3B-negative cells were shrunken or fragmented, indicating the presence of typical apoptotic nuclei. By contrast, the nuclei of TUNEL- and LC3B- double positive cells were round, as usually observed in autophagic cell death (19) (Fig. 6).