Methods
Animal experimentation was performed at the Life and Health Sciences Research Institute at the University of Minho, in accordance with the Directive 2010/63/EU, and approved by Institutional Animal Care and Use Committee of University of Minho. 8–12-week-old male C57BL/6 mice (Charles River Laboratories) were housed under specific pathogen-free condition with food and water ad libitum and acclimatized for 1 week before the experiment. Mice (n = 14) were equally and randomly divided in two groups corresponding to established endpoints of 2- and 9-days post-infection (dpi). Randomization was performed using randomize function of Microsoft® Excel®. Humane endpoints were followed as described on Additional file 1: Table S1.
Diabetes induction
Type 1 diabetes mellitus (T1DM) was chemically induced, as previously described [11], administering 50 mg/kg of streptozotocin (STZ) (Merck KGaA, Germany) for 5 consecutive days. Blood glucose levels were measured 9 days after STZ treatment, using a monitor glucose device. Levels of blood glucose higher than 150 mg/dL were considered hyperglycemic. Mice were observed for signs of polydipsia and polyuria throughout the experimental period.
Dorsal fur depilation
On the day before surgery, dorsal fur of mice was shaved, using a hair clipper followed by depilatory cream for 1 min. Cream was further removed by wiping the skin with cotton soaked in warm water.
Inoculation of polycarbonate membranes
Briefly, 0.2 µm pore size polycarbonate membranes (Merck KGaA, Germany) were cut in 5-mm diameter discs, sterilized on both sides by UV light for 30 min and then placed on mannitol salt agar (MSA). A bacterial suspension of 108 colony forming units (CFU)/ml of S. aureus Rosenbach (ATCC BAA 2313) was prepared using isolated colonies previously grown on MSA, that were resuspended in saline and further inoculated on the membranes (102 CFU/membrane). Inoculated membranes were incubated overnight at 37 ºC to grow a biofilm (attaining approximately 109 CFU/membrane).
Excisional wounding and infection
Mice were intraperitoneally injected with anesthetics (75 mg/kg ketamine and 1 mg/kg medetomidine) and analgesic (0.1 mg/kg buprenorphine). Two symmetrical full-thickness excisional wounds were created using a 5-mm diameter punch biopsy, by placing mice on their side, pulling the dorsal skin and perforating through the folded skin (Fig. 1A). To minimize wound contraction, a silicone splint ring (15-mm external and 6-mm internal diameter) was positioned around each wound using cyanoacrylate glue and secured with four interrupted sutures of 5/0 nylon (Fig. 1B). During the procedure, wounds were maintained hydrated with saline.
Wounds were infected by placing the S. aureus-inoculated polycarbonate membrane face down for direct contact of biofilm with the wound bed (Fig. 1C). Finally, wounds were covered with Durapore™ self-adhering bandage (3 M, USA) (Fig. 1D). Mice received 1 mg/kg atipamezole intramuscularly to revert the effect of anesthesia and were then placed under a warming lamp until full recovery. For postoperative pain relief, analgesia was administered subcutaneously with maximal intervals of 12 h during the two following days. Mice also received a vitamin supplementation (Duphalyte®) by subcutaneous injection, to avoid massive weight loss and dehydration.
Two days after wounding/infection, a biofilm covering the wounds was observed (Fig. 1E). At this point, mice received a light sedative (7.5 mg/kg ketamine and 1 mg/kg medetomidine, intraperitoneally) to remove the polycarbonate membranes. Topical treatment can be applied at this point, if desired. A sterile transparent semi-occlusive dressing Tegaderm (3 M, USA) was then applied covering wounds and splints, followed by an Omnifix elastic bandage (Hartmann) (Fig. 1F).
Wound tissue analysis
To assess the validity of the proposed model, wound tissue was collected for microbiological and histological analysis at 2- and 9-dpi. Mice were sacrificed with an overdose of isoflurane, and dressings and splints were carefully removed. Using a scalpel blade, wounds and surrounding skin tissue were harvested. One wound was used for bacterial burden quantification, while the other was dissected for histological analysis. For bacterial quantification, wound tissue was minced, tenfold serially diluted in sterile saline and cultured on MSA at 37 ºC/24 h. The number of viable bacteria was expressed as Log10CFU/wound. For histological analysis, tissue was divided across the wound center and immersed in neutral buffered formaldehyde (4%, w/v), embedded in paraffin, and then sectioned in samples of 4-µm thickness for staining with Hematoxylin and Eosin (H&E) and Gram.
Statistical analysis
Data were reported as mean ± standard deviation. Differences were assessed through Student’s t-test, using Graphpad Prism 7 (Graphpad Software Inc., CA). Statistical significance was set at p < 0.05.