Our study was performed in accordance to the Recommendations of Guidelines for Clinical Trials by the Ethics Committee of Nanjing Drum Tower Hospital. Adhesive peritoneum tissues were obtained from ASBO patients during laparotomy or laparoscopy (Supplementary Fig.1a). Informed consent was obtained from all subjects after surgery.
Adhesion-induction surgery was performed as previously described . In brief, a small portion of the peritoneum was grasped and ligated at its base using 4-0 silk suture (Ethicon), creating a standardized peritoneal button. This was repeated for a total of four buttons (one in each quadrant). Light abrasion on each button and on the adjacent liver, cecum, and intestine was performed using surgical brash. Finally, peritoneum and skin were closed using 4-0 silk suture. Mouse ischemic button model is easily reproducible to induce intra-abdominal adhesions compared to other models as previously demonstrated . The experimental procedure in this study was approved by the Animal Care and Use Committee of Nanjing Drum Tower Hospital.
Histology, IHC and immunofluorescence
Tissues were fixed in 2% PFA overnight at 4°C and were embedded in paraffin. paraffin sections were cut at 5μm. H&E, Masson’s trichome stains, and immunohistochemistry (IHC) were performed following standard protocols.
For IHC staining, slides were incubated with anti-APD4 antibody, anti-citH3 antibody, and anti-α-SMA antibodies according to the manufacture’s instruction. Nuclei were stained with DAPI (Invitrogen).
Immunofluorescence experiment was performed on paraffin sections. NETs associated proteins were evaluated by immunofluorescent staining using rabbit mouse anti-mouse MPO, and rabbit anti-mouse citH3 (Abcam, Cambridge, UK). DAPI was use to counterstain DNA. Images were acquired using a Leica DMI 4000 B fluorescence microscope.
Enzyme-linked immunosorbent assays (ELISA) was performed to evaluate the production of inflammatory cytokines, including IL-1β, IL-6, and TNF-α according to the manufacturer’s instructions.
Mice were injected with 100 µg intraperitoneal (i.p.) monoclonal anti-mouse Ly6G at 12h or 3d after induction of ischemic button. Rat IgG2a isotype control was administered in the same way. To verify neutrophil depletion, blood neutrophil levels were evaluated by flow cytometry and analyzed using FlowJo V10 software as previously described .
DNase I and PAD inhibitor administration
Mice received i.p. injection of 50 μg DNase I at 2h, 12 h or 3d after induction of ischemic button. Control counterparts were injected with PBS. PAD inhibitor Cl-amidine was dissolved in dimethyl sulfoxide (DMSO) as previously described. The stock solution was dissolved in saline and injected i.p. (10 mg/kg) at 2h, 12h and 3d after adhesion induction. Vehicle (DMSO) was administered as control (Fig.3a).
The peritoneal ischemic buttons were dissected after the mice were sacrificed, and the protein from adhesive tissues and HPMCs were extracted using RIPA lysis buffer. Proteins were separated by 10% SDS-PAGE, and transferred to PVDF membrane. After blocking with 5% milk, the membranes were incubated with anti-Ly6G, anti-PAD4, anti-citH3, anti-p-TBK1 and anti-p-IRF3 at 4 °C overnight. GAPDH served as internal control.
All statistical analyses were evaluated using SPSS 19.0 and GraphPad Prism 6. All data were presented as mean ± SEM. Unpaired t test and one-way analysis were used to compare continuous variables between groups. P<0.05 was considered significant.