Betadine

Mice underwent either sham surgery or focal cerebral ischemia as previously described (Evans et al. 2017 ). Cerebral ischemia was produced in anesthetized mice (ketamine: 80 mg/kg plus xylazine: 10 mg/kg i.p.) by occlusion of the middle cerebral artery (MCA) using a 6.0 silicone-coated monofilament (Doccol Corporation). Rectal temperature was monitored and maintained at 37.0 ± 0.5 °C. MCA occlusion (MCAO) was sustained for 60 min, and the filament then retracted to allow reperfusion. Both successful occlusion (> 70% reduction in cerebral blood flow; CBF) and reperfusion (≥ 80% return of CBF to the pre-ischemic level) were confirmed by transcranial laser-Doppler flowmetry (PeriMed). Sham-operated mice were anesthetized and the right carotid bifurcation exposed, but no filament was inserted. Neck wounds were then closed with sutures and covered with Betadine^® (Sanofi) and spray dressing. Head wounds were closed with superglue, and mice were returned to their cages after regaining consciousness.

A head biopsy model was employed, as recommended for assessment of growth factors in wound healing [18 (link),19 ]. Briefly, five female BALB/c mice per group (rTRX, PBS and rOv-GRN-1) were anesthetized (intraperitoneal xylazine 16 mg kg^-1; ketamine 80 mg kg^-1), after which the crown of the head was shaved with an electric razor. Mice were anesthetized three days later and the surgical site was sterilized with 70% ethanol wipes. A skin-deep wound of 5 mm in diameter was inflicted on the crown of the head using biopsy punch (Zivic instruments). The lesion was rinsed with antiseptic (Betadine, Sanofi), after which 56 pMoles of rOv-GRN-1, rTRX or PBS suspended in 1.5% methyl cellulose (Sigma) in 50 μl was applied. Thereafter, the lesion was covered with Elastoplast Spray Plaster (Beiersdorf). Progress of the wound, and wound closure, was documented with photographs taken at cumulative 1.6× magnification using a dissection microscope (Olympus) fitted with a Nikon D200 camera, each day for five days. Wound closure was ascertained in an unblinded fashion by comparison of the surface area of the lesion with the size as documented immediately after the wound was inflicted, with the assistance of ImageJ software.