Acute peritoneal adhesion rat models
This study was approved by the Ethics Committee of The General Hospital of the People's Liberation Army (Permit Number: 2010-X-3-28) with animal care performed strictly according to established institutional guidelines. All surgery was performed under pentobarbital anesthesia. Scrape-induced peritoneal adhesions were created in healthy male Sprague-Dawley (SD) rats weighing 200 g to 250 g. All animals were obtained from the Experimental Animal Center of the Academy of Military Medical Sciences (Beijing, China) and housed at constant room temperature with a 12-hour light/dark cycle. Standard rodent chow and water were provided ad libitum. The animals were acclimated for seven days before initiating the experiment.
Surgical procedures were conducted by a single surgeon under aseptic conditions in the Laboratory Animal Unit. Rats were anesthetized with a 2% pentobarbital (30 mg/kg) intraperitoneal injection. Briefly, a 2-cm vertical midline incision was made into the abdominal wall and peritoneum. The dorsal and ventral surfaces of the cecum were scraped with dry gauze 20 times over an area of 2 × 2 cm2 until petechial bleeding occurred, and the cecum was then replaced. The parietal peritoneum lateral to the midline incision was scraped 20 times until petechial bleeding occurred. The incision was closed in two layers with 4/0 silk sutures . After surgery, the rats were kept in a single cage and fed a normal diet.
SD rat bone marrow-derived MSCs/red fluorescent protein (RFP) was obtained commercially (Cyagen Biosciences, Sunnyvale, CA, USA). The culture was initiated following the manufacturer's instructions. MSCs were placed into 25 cm2 culture flasks and cultured with MSCs growth medium (Cyagen Biosciences, Sunnyvale, CA, USA) at 37°C under 5% CO2 and 90% humidity. The medium was changed every three days. Sixth to eighth passage MSCs were used for the experiments. Following previous methods , fluorescence-activated cell sorting (FACS) analysis (Beckman Coulter, Indianapolis, IN, USA) was used to examine the representative markers of MSCs (CD45, CD90 (BD Biosciences, San Diego, California, USA); CD11a, CD54 (AbD Serotec, Oxford, UK)), and multilineage differentiation of MSCs was examined under adipogenic and osteogenic differentiation conditions.
Transfection of MSCs with TSG-6 small interfering RNA (siRNA)
Fifty-percent confluent MSCs were transfected with 20 nM TSG-6-small interfering (si) RNA or the siRNA-negative control (NC) (GenePharma, Shanghai, China) using INTERFERin™(Polyplus-transfection SA, Bioparc, France). At 24 hours after transfection, MSCs were fed with serum-free medium for 24 hours, prior to experiments. To confirm the knockdown of TSG-6, RNA was assayed for TSG-6 by RT-PCR (TSG-6 forward primer: AGTGATGCGTCCGTCACAGCC, reverse primer: AGATGGCTAAACCGTCCAGCTAAGA, product length = 134 bp; GAPDH forward primer: GGCATGGACTGTGGTCATGAG, reverse primer: TGCACCACCAACTGCTTAGC, product length = 87 bp (SBS Genetech, Beijing, China)), and the protein was assayed for TSG-6 by Western blot (primary antibodies TSG-6 (1:50) (Santa Cruz Biotechnology, Santa Cruz, CA, USA)).
Injection of MSCs or recombinant mouse (rm) TSG-6
At 0, 4, 12, 24, or 48 hours after peritoneal scraping, MSCs (5 × 106) in 1-ml serum-free medium were injected via the tail vein or peritoneum. At 24 hours after peritoneal scraping, TSG-6-siRNA-MSCs, TSG-6-siRNA-NC-MSCs or 3 ng/ml rmTSG-6 (97% homology with rat ) (R&D Systems Inc., Minneapolis, MN, USA) in 1-ml serum-free medium (adapted from our previous experiment ) were injected via the tail vein. Rats injected with the serum-free medium were the negative control, and the rats without peritoneal scraping were the blank control.
Two-photon fluorescence confocal imaging of MSCs after injection
A Leica two-photon fluorescence confocal imaging TCS SP5 system (Leica Microsystems, Mannheim, Germany) was used to evaluate the distribution of MSCs after injection. The excitation and emission filter set for green (autofluorescence) detection was 488 nm and 504 to 569 nm, respectively. The excitation and emission filter set for red detection was 543 nm and 555 to 624 nm, respectively. MSCs were injected into rats intraperitoneally or intravenously at 24 hours after scraping. Rats were sacrificed at 4, 12, 24, 48, and 72 hours and 5, 7 days thereafter (n = 3 in each group at each time point). Fresh thick tissues of the right lung, right lower liver, spleen, and scraped peritoneum were sampled.
Immunofluorescence staining of lung and spleen
MSCs, TSG-6-siRNA-MSCs, or TSG-6-siRNA-NC-MSCs were injected into rats intraperitoneally or intravenously at 24 hours after scraping. Rats were sacrificed at 4, 12, 24, 48 and 72 hours and 5, 7 days thereafter (n = 3 in each group at each time point). The right lung and the spleen were sampled. Specimens were embedded in optimum cutting temperature (OCT) compound and stored at -80°C until use. Frozen tissues were sectioned every 4 μm and placed on poly-L-lysine precoated slides. The slides were fixed with 4% paraformaldehyde for 5 minutes at room temperature, and for 10 minutes at 4°C. The slides were then blocked with 1% BSA for 30 minutes at room temperature. The following primary antibodies were incubated overnight at 4°C: ED-1 (1:50) (Santa Cruz Biotechnology) and TSG-6 (1:50) (Santa Cruz Biotechnology, sc-30140). Secondary antibodies conjugated with fluorescein isothiocyanate (FITC) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were applied for 1 hour at room temperature in a darkened humidified chamber. Finally, the preparations were mounted in fluorescent mounting medium with 4',6-diamidino-2-phenylindole (DAPI). Negative controls did not receive the first antibody. Each tissue section was observed under a confocal laser scanning microscope (Olympus FluoView 1000, Tokyo, Japan) at magnifications of × 600 and × 1800. Three-dimensional imaging was applied, if necessary.
Enzyme-linked immunosorbent assay (ELISA) of TSG-6 in rat serum
Rats were sacrificed at 4, 12, 24, 48, or 72 hours, or 5, 7, 14 days after MSCs, TSG-6-siRNA-MSCs, or TSG-6-siRNA-NC-MSCs injection intravenously at 24 hours after scraping (n = 3 in each group at each time point). Quantification of TSG-6 in serum was performed by ELISA according to the conventional procedure. Absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). TSG-6 concentrations were determined with a standard curve constructed by titrating rmTSG-6. All samples were placed in three replicate wells. A laboratory-made ELISA kit was prepared mainly as follows: a 96-well microplate (Corning, Lowell, MA, USA) was coated with mouse monoclonal antibody to TSG-6 (Santa Cruz Biotechnology, sc-377277), enzyme-labeled secondary antibody was purchased from Dako (Glostrup, Denmark). The correlation coefficiency was 0.995 for the standard curves.
Macroscopic evaluation of peritoneal adhesions
At 0, 4, 12, 24, or 48 hours after peritoneal scraping, MSCs were injected intraperitoneally. At 24 hours after scraping, MSCs or rmTSG-6 were injected intravenously. Rats were sacrificed on day 14 after scraping (n = 6 in each group at each time point). The size and severity of peritoneal adhesions were evaluated macroscopically by an independent observer on a scale of 0 to 4 (0, 0%; 1, <25%; 2, 25% to 49%; 3, 50% to 74%; and 4, 75% to 100% adhesions) using a previously reported scoring system .
Histological analysis of peritoneal adhesions
Rats were sacrificed on day 14 after scraping (n = 6 in each group at each time point). The entire fibrous band was sampled. Specimens were fixed in 10% formaldehyde for 24 hours. After dehydration, they were embedded in paraffin, and 3-μm thick cross-sections were stained with Masson's trichrome. Each tissue section was examined by light microscopy (Olympus IX71, Tokyo, Japan) at magnifications of × 100 and × 400. Five randomly selected fields in each section were evaluated by an independent pathologist (at a magnification of × 100). The extent of fibrosis was scored as 0 (negative), 1 (weak), 2 (medium), or 3 (intensive).
Analysis was done using SPSS Statistics, version 17.0.2. Results are presented as mean values ± standard deviations. Multiple comparisons of parametric data were performed using one-way analysis of variance (ANOVA). Nonparametric data were compared with the Mann-Whitney U-test to identify differences between groups. A value of P <0.05 was considered to indicate statistical significance.