EPR Method for ex vivo Detection of Reactive Oxygen Species in Tissue

Abstract

Measurement of free radicals, ex vivo, in biological tissue with continuous wave electron paramagnetic resonance (EPR) has been problematic. Here, a novel EPR spin trapping technique has been developed that places one or two large segments of porcine arterial tissue along with supernatant directly into a standard EPR spectrometer cavity at liquid nitrogen temperature. This approach is validated in porcine arterial tissues treated as controls in vivo. To investigate that this method has lower extraneous ROS generation than methods which slice tissue into thin slivers before assaying for reactive oxygen species (ROS), we take two sets of coronary arterial tissue from the same animal. In the first set, we excise one or two large segments of tissue, and in the second set we section the tissue into ten pieces. In both cases, tissue was harvested, placed in physiological saline solution with 190-mM PBN (N-tert-butyl-α-phenylnitrone) and incubated in the dark for 2 hours at physiological temperature while gently being stirred. Tissue and supernatant were then loaded into a syringe and frozen at minus eighty degrees Celsius until EPR analysis. Although absorption of microwaves by an aqueous sample typically at room temperature limits the size of the sample; this method can accommodate as large as 20-mm3 tissue volume into the EPR cavity at liquid nitrogen temperature. In our experiments the tissue volumes were in a range of values between 2 mm3 and 5 mm3,and the EPR spectra were normalized with respect to tissue volume.