023 HRE-Luciferase Transfection Quantifies Tissue Level Ischemia in a Rabbit Ear Wound Model

Abstract

The Hypoxia Inducible Factor (HIF) system has been characterized as the principal tissue level response to hypoxia. Posttranslational regulation of HIF-1alpha has been reported to act though a Hypoxia Responsive Element (HRE) promoter region on a range of hypoxia-induced genes. A plasmid was constructed consisting of a fivefold HRE repeat conjugated to a luciferase gene, used as a marker for HRE activation. Plasmid HRE-luciferase was then transfected into a well-established ischemic rabbit ear wound healing model. Ischemia was induced using a variety of published models for interruption of arterial inflow and compared with a nonischemic control. Central artery, caudal artery and rostrocaudal artery models were tested for induction of ischemia. Tissue specimens were harvested from the transfected areas and solubilized. Luminescence of each specimen was measured using a standard luminometer to quantify luciferase induction. To provide correlation on a regional level, tissue level oxygen tension was measured directly for each wound model. Profound and statistically significant differences were found in the induced luciferase production. Marked differences in the tissue hypoxia between the various ischemic wound healing models correlated in graded fashion with the ischemia induced on an anatomic basis. The least ischemic model showed no significant difference in hypoxia readings versus control. The intermediate model showed a significant fourfold greater ischemia signaling. The most ischemic model showed a highly significant 72-fold greater ischemic response compared to controls. The use of gene transfection is described as a sensitive and effective method for quantification of tissue hypoxia at a cellular level in ischemic wounds. Acknowledgments: This study was funded by the Wound Healing Research Laboratory.