Rat cutaneous MRSA wound infection model

All animal experiments and procedures were approved by Hubei Provincial Center for Disease Prevention and Control, and the Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. All animals were kept and utilized in accordance with the Animal Management Rules of the Ministry of Health of the People’s Republic of China and the Guidelines for the Care and Use of Laboratory Animals of China. Eight-week-old Sprague–Dawley male rats (~200 g) were obtained from the Hubei Provincial Center for Disease Prevention and Control. A total of 120 Sprague–Dawley male rats were randomly divided into the following groups: control, ZnPB-3, Van, Fus, Ret and Mup. First, the rats were anaesthetized by intraperitoneal injection of 3% pentobarbital (1 mL kg⁻¹) prior to surgery and placed on a sterile drape to provide sterile conditions during surgery. The dorsal side of the rats was shaved, depilated and disinfected. Two symmetrical round wounds with 6 mm in diameter were made on the dorsum of each rat using a biopsy punch. Then, each wound from each group was infected with a MRSA dose of 10⁷ CFU per wound, and treated with PBS (control) and ZnPB-3 (0.2 mg kg⁻¹) with 808 nm laser irradiation at a power density of 0.3 W cm⁻² for 15 min. Afterwards, the treated wounds were covered with nonwoven fabrics and fixed with surgical adhesive. Comparatively, the treatment with Van is set as the positive control for systemic antibiotic therapy of MRSA-infected wounds using different dosages (10, 40 and 160 mg kg⁻¹ day⁻¹) through intravenous injection, and the treatments with Van, Fus, Ret and Mup are also set as positive controls for topical antibiotic therapy of MRSA-infected wounds using the same dosage (0.2 mg kg⁻¹ day⁻¹).

To evaluate the therapeutic effect of ZnPB for MRSA wound infection, the bacterial counts in each wound were assessed by the spread plate method at each time point (days 1 and 2), and the wounds were observed and photographed at regular time intervals. For histomorphological analysis, the rats were sacrificed, and the skin tissues of the wounds were harvested and fixed with 4% paraformaldehyde solution and then embedded in paraffin. The samples were stained with H&E staining on days 2, 4, 8 and 12, Masson’s trichrome staining on day 12 and Sirius red staining on day 12. To evaluate in vivo biosafety, the major organs (heart, liver, spleen, lung and kidney) were stained with H&E on day 12.