Page 269 - Prevention and Treatment of Incisional Hernia- New Techniques and Materials

Macrophages in a contaminated environment
Methods
Rat peritonitis model and tissue collection
The protocol of the rat experiment was approved by the Ethical Committee on Animal Experimentation of Erasmus University Rotterdam, The Netherlands, and is in accordance with the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines. A contaminated environment was created by the caecal ligature puncture model, in which the caecum is punctured to provide leakage of faecal uid into the abdominal cavity, thus causing peritonitis. After 24 h the abdominal cavity was re-opened, peritonitis was con rmed by microbiological culture, and a mono lament polypropylene (PP) biomaterial (ParieteneTM; Covidien – Sofradim Production, Trévoux, France) was placed intraperitoneally in four rats(15). Some 28 days after implantation, a sample of the abdominal wall with the incorporated biomaterial was harvested using biopsy punches (5 mm diameter). As controls, abdominal walls from rats with peritonitis, but with no biomaterial, were collected. All tissue samples were xed in 4 per cent formalin and embedded in para n.
Histology and immunohistochemistry
Tissue sections were cut and stained with haematoxylin and eosin in accordance with standard procedures. To identify macrophage types, immunohistochemical staining with the following antibodies were used; CD68, a general macrophage marker; CD206, a marker for M2 macrophages(8), and inducible nitric oxide synthase (iNOS) as a marker for M1 macrophages(10). Brie y, para n sections were dewaxed and, to block the sections for aspeci c binding, the sections were pretreated with heat-mediated antigen retrieval solution (Target Retrieval Solution; Dako, Glostrup, Denmark) at 90°C for 20 min. Sections were incubated with CD68 (1 : 100; Acris, Herford, Germany), CD206 (1 : 100) or iNOS (1 : 50) (both Abcam, Cambridge, UK) for 60 min, and subsequently incubated with link and label (Concentrated MultiLink® and Concentrated HRP Label (peroxidase-conjugated streptavidin); BioGenex, Fremont, California, USA); 3,3′-diaminobenzidine was used as substrate. Sections were dried overnight and mounted with VectaMountTM (Vector Laboratories, Burlingame, California, USA). Matching irrelevant isotype antibodies were used as negative controls, and tissues known to contain the speci c markers were employed as positive controls. Sections were also Gram-stained to visualize potential bacteria. All
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