Strategies for Development of Chronic Wounds Using the LIGHT-/- Mouse and a Diabetic Mouse: From Mechanism to Treatment
Wound healing involves many cellular and molecular processes that are integrated in several sequential and overlapping phases, hemostasis, controlled oxidative stress, inflammation, granulation tissue formation, and remodeling. Impaired-healing and chronic wounds exhibit defective regulation of one or more of these processes that leads to conditions such as diabetic foot ulcers, and other similar chronic wounds that impact ~6.5M people and cost ~$25B/year in the US alone. Great efforts have been made to stimulate healing of these wounds, including the development of animal models mimicking chronic wounds in order to understand how they develop but success has been limited. Recently, we developed a mouse model of impaired healing that became chronic in presence of biofilm-forming bacteria. I took an integrated approach by using various cellular and molecular approaches to study the wound microenvironment. Using the LIGHT-/- model of impaired healing, I showed that the wounds in these mice, very early during the process of healing have elevated levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), increased lactate levels and reduced pH that could potentially damage the healing tissue. With the use of luminol I showed, for the very first time, real time monitoring of increase in oxidative stress levels. In addition, I showed that the detrimental effects of increases in ROS and RNS significantly increased damage to DNA, lipid peroxidation and protein nitrosylation. Furthermore, using a lipidomics approach I showed an increase in inflammatory lipids and lipids involved in platelet function. The findings were confirmed by the increases in inflammation, platelet aggregation and reduced bleeding time post wounding. I then showed that by exacerbating the levels of ROS by inhibition major antioxidant enzymes, glutathione peroxidase and catalase, and introducing previously isolated biofilm forming bacteria on the wound bed, led to the development of chronic wounds. The wounds remained open and persistent inflammation was marked by the clear presence of neutrophils and macrophages in the wound tissue. The granulation tissue was poorly formed and there was loss of collagen bundles. Furthermore, I also showed that the bacteria were capable of forming biofilms and were resistant to antibiotics. These results confirmed that redox imbalance and presence of bacteria were crucial elements for chronic wound formation.
I then tested the possibility that exacerbated oxidative stress was critical for chronic wound development by performing similar experiment in a diabetic mouse model, the db/db. I showed that only one dose of inhibitors to the antioxidant enzymes at the time of wounding was sufficient to cause the wound to become chronic by 20 days and spontaneously harbor biofilm-forming bacteria. The chronic wounds in these mice did not heal for as long as 90 days. I also showed that the bacteria was resistant to antibiotics and that there were embedded in the extrapolysaccharide (EPS) matrix. To confirm the importance of redox stress, I reversed the stress levels by treating the wounds with antioxidants, N-acetyl cysteine and α-tocopherol, and showed that the wounds healed with decreased levels of oxidative stress, reduction in biofilm forming bacteria, reduction in biofilm on the wound bed, better tissue structure with proper collagen bundles and proper differentiating epithelial cells.
The studies presented here provide an in vivo model of chronic wounds that captures many of the clinical aspects of human chronic wounds and that may provide insights into the mechanisms involved in chronic wound development, including the natural growth of biofilm in situ. These findings, taken together suggest the robustness of the LIGHT-/- and db/db models of impaired and chronic wounds respectively that warrants further research to capture the underlying mechanisms in the development of human chronic wounds and hence uncovering potential targets to treat such wounds.