Impact of MSCs against Hepatic I/R Injury Figure 8. A new rat model of major hepatectomy with I/R injury. doi:10.1371/journal.pone.0019195.g008 the animals were allowed to awaken and then given free access to food and water. Animals were sacrificed after the reperfusion of 6, 24, 72, and 168 h (n = 6 per each point). Blood samples were taken from the inferior vena cava for liver function tests, and the liver was harvested and weighed. Liver tissue samples were collected and properly preserved for subsequent procedures. Immunofluorescence Cells were fixed in 4% formaldehyde for 10 min, followed by incubation with Protein Block for 30 min. Bone marrow-derived MSCs were analyzed by immunohistochemistry using monoclonal anti CD29 (VMRD, Inc., USA), CD31 (santa cruz biotechnolog, Inc., USA), CD34 (R&D Systems, USA), and CD105 (santa cruz biotechnology, USA) antibodies overnight at 4uC [22,23]. The fluorescein (green, 1:3000)-conjugated secondary antibody (COSMO BIO, Inc., Tokyo) against CD31, CD34, and CD105 was applied for 30 min. Rhodamine (red, 1:3000)-conjugated secondary antibody (Rockland immunochemicals, Inc., Tokyo) against CD29 was also used. Evaluation of the mesenchymal lineage differentiation of MSCs The differentiation potential of MSCs (passage 5) into adipocytes, osteocytes, or chondrocytes was evaluated using differentiation-induction media purchased from Lonza Walkers- ville, Inc. (http://www.lonza.com) according the manufacturer’s protocols. Detection and quantification of transgene expression using a non-invasive in vivo imaging system In Vivo Imaging System IVISTM (Xenogen, Allameda, CA) was used for the analysis of luciferase gene expression activity. In this system, a noninvasive charged-couple device camera was used to detect bioluminescene emitted from D-luciferin, which reacts with firefly luciferase in living animals. While under isoflurane anesthesia, the rats received D-luciferin through the penile vein [30 mg/kg/body weight, dissolved and diluted to 15 mg/ml in PBS] [24]. Immediately after the infusion, the light emitted by luciferase was measured, with a 1-min integration time. The signal intensity was quantified as photon flux in units of photons/sec/ cm2/steradian in the region of interest [25]. The results show the mean values of 3 rats in each of 6, 24, 72, 120, and 168 h after reperfusion. Assessment of liver functions Blood samples were obtained from each rat and centrifuged for 10 min at 3,000 rpm, and serum was collected. Concentration of markers of liver injury such as GOT and GPT was analyzed using a FUJIFILM DRI-CHEM 3500 machine (FujiFilm, Tokyo, Japan; http://www.fujifilm.co.jp) and FUJI DRY CHEM SLIDES (FujiFilm), respectively, for GOT/asparatate aminotransferase (AST)-PIII and GPT/alanine transaminase (ALT)-PIII. Histological analysis After euthanasia, livers were fixed with 10% buffered formalin for paraffin embedding, or in OCT compound for frozen sections with no fixation. Five-micron paraffine-embedded sections were stained with hematoxyline and eosin (H&E) for conventional morphological evaluation. Suzuki classification [26], which consisted of 3 parameters of hepatic ischemia reperfusion injury: sinusoidal congestion, vacuolization of hepatocyte cytoplasm, and parenchymal necrosis. Each parameter was graded numerically as follows: congestion: 0 = none, 1 = minimal, 2 = mild, 3 = moder- ate, and 4 = severe. The same criteria were utilized in the graduation of the vacuolization, and for necrosis, the numerical graduation was as follows: 0 = nonnecrotic cells, 1 = single cell PLoS ONE | www.plosone.org 7 April 2011 | Volume 6 | Issue 4 | e19195