16.0: Thursday, March 17, 2005

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

Hakim K. Said, MD, John Y. S. Kim, Nakshatra Roy, and Thomas A. Mustoe.

The Hypoxia Inducible Factor (HIF) system has been characterized as the principal tissue level response to hypoxia. Post-translational 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 five-fold 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 non-ischemic control. Luminescence of each harvested specimen was measured using a standard luminometer to quantify luciferase induction as an indicator of ischemia signaling. For regional correlation, tissue level oxygen tension was measured directly for each wound model. Marked differences in the tissue hypoxia between the various ischemic models correlated in graded fashion with the ischemia induced on an anatomic basis. The intermediate and profound ischemia models showed significant 4 fold and 72 fold greater ischemia signaling respectively versus 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.