Endogenous Toll-like Receptor 4 Deletion Significantly Improves Wound Healing in a Murine Model of Diabetic-ischaemic Ulceration

Abstract

Introduction: Diabetic foot ulceration is a multifactorial, common and challenging complication of diabetes. Between 15% and 25% of diabetics develop ulceration in their lifetime, leading to a 20 times greater risk of major amputation. There is increasing recognition that toll-like receptor 4 (TLR4) mediated inflammation is involved in the systemic pathogenesis of diabetes and may contribute to impairment of wound healing. We have previously shown in vitro that hyperglycaemia and ischaemia act synergistically to impair fibroblast function and survival via the pro-inflammatory MyD88-dependent pro-inflammatory TLR4 pathway. Here, we aim to study the effect of TLR4 knock-out (KO) on wound healing in a murine diabetic-ischaemic model of hindlimb ulceration. Methods: Diabetes was induced in wild-type (WT) and TLR4 KO C57BL/6 male mice by intra-peritoneal injection of low-dose streptozocin (STZ 50mg/Kg/day for 5 days) and was confirmed at day 14 by tail capillary blood sampling (BM of >16mmol/l). Glycosuria was monitored with weekly urinalysis. Hindlimb ischaemia was induced by femoral artery ligation at four weeks post STZ, and a full thickness 4mm skin wound inflicted below the knee. Wound healing was assessed via digital planimetry at days 3, 7 and 14 post surgery. Results: Diabetic-ischaemic wounds demonstrated significantly impaired wound healing compared to diabetic non-ischaemic, ischaemic non-diabetic or non-diabetic, non-ischaemic wounds at day 14 (p < 0.05). There was no significant difference in healing rate between diabetes only and ischaemia only wounds. Diabetic-ischaemic wounds in TLR4 KO mice demonstrated significantly improved healing rates compared to those in WT mice at days 7 and 14. 67% of wounds were completely healed in TLR4 KO mice compared to 0% in WT mice by day 14. Conclusion: Our in vivo model demonstrated significantly impaired wound healing in diabetic-ischaemic animals. In WT animals, no hind-limb wound had completely healed at day 14, suggesting this is a successful model of chronic diabetic ulceration. In TLR4 KO mice, a significant and dramatic improvement in the healing of diabetic-ischaemic wounds was observed. We conclude that endogenous TLR4 deletion confers a beneficial effect on wound healing in diabetic-ischaemic conditions in vivo, and suggests a potential therapeutic role for TLR4 antagonism in chronic diabetic ulceration.