Quantifying tissue level ischemia: hypoxia response element-luciferase transfection in a rabbit ear model.
Ischemia is a common underlying factor in a number of pathologic conditions ranging from cardiac dysfunction to delayed wound healing. Previous efforts have shown the resulting hypoxia activates the hypoxia inducible factor, a transcription factor with signaling effects through an intranuclear hypoxia response element (HRE). We hypothesized that ischemic conditions should activate these hypoxic signaling pathways in a measurable manner. We tested our hypothesis using variations of an established rabbit ear ischemic wound model and an HRE-luciferase-reporter gene construct. This plasmid construct was transfected into the ears of young, female New Zealand White rabbits, harvested at day 7 and processed to yield a reactive solution. Luminometry was used to quantify luciferase expression in each solution as a marker for HRE activation in each wound. Quantitative readings of hypoxic signaling as measured by luminescence yielded profound and statistically significant differences between the various ischemic models. Our results suggest that the biologic systems for hypoxic signaling can be used to detect local ischemia. HRE-luciferase transfection is an effective tool for quantifying the degree of tissue hypoxia. The caudal ischemic rabbit ear model showed significantly higher levels of hypoxia. Use of a validated model that produces sufficient tissue levels of hypoxia is recommended for meaningful study of ischemic wound healing.
Said
Hakim K
HK
Division of Plastic Surgery, University of Washington, Seattle, Washington, USA.
Roy
Nakshatra K
NK
Gurjala
Anandev N
AN
Mustoe
Thomas A
TA
D000818:Animals; D015687:Cell Hypoxia / Q000235:genetics* / Q000502:physiology; D004423:Ear / Q000098:blood supply / Q000293:injuries; D005260:Female; D007511:Ischemia / Q000235:genetics* / Q000503:physiopathology; D008156:Luciferases / Q000235:genetics*; D023421:Models, Animal; D011817:Rabbits; D020218:Response Elements / Q000235:genetics*; D012720:Severity of Illness Index; D012867:Skin / Q000098:blood supply / Q000293:injuries; D014162:Transfection; D014945:Wound Healing / Q000502:physiology
D008156:Luciferases