In Vivo Immuno-Spin Trapping: Imaging the Footprints of Oxidative Stress

Abstract

A plethora of disease processes are associated with elevated reactive species formation and allied reactions with biomolecules that alter cell signaling, induce overt damage, and promote dysfunction of tissues. Unfortunately, effective detection of reactive species in tissues is wrought with issues that significantly limit capacity for validating species identity, establishing accurate concentrations, and identifying anatomic sites of production. These shortcomings reveal the pressing need for new approaches to more precisely assess reactive species generation in vivo. Herein, we describe an in vivo immuno-spin trapping method for indirectly assessing oxidant levels by detecting free radicals resulting from reaction of oxidants with biomolecules to form stable, immunologically detectable nitrone-biomolecular adducts. This process couples the reactivity and sensitivity of an electron paramagnetic resonance spin trap with the resolution of confocal imaging to visualize the extent of cell and tissue oxidation and anatomic sites of production by detecting resultant free radical formation.

Keywords: 5,5-dimethyl-1-pyrroline N-oxide (DMPO); electron paramagnetic resonance (EPR); free radicals; immuno-spin trapping; oxidative stress; reactive species.

Figure 12.42.1

Immuno-spin trapping of biomolecular free radicals in pulmonary tissue of obese mice. (Top) The reactions involved in the immuno-spin trapping (IST) process. (Bottom) A representative image of an airway and surrounding tissue that was generated by exposing a diet-induced obese mouse (C57Blk/6 J, subjected to a diet consisting of 60% calories derived from fat for 20 weeks) to 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The presence of abundant DMPOadducted biomolecules (red) is seen in the pseudostratified ciliated columnar epithelial cells of the airway as well as the basolateral space, alveoli, and interstitium. This is indicative of elevated reactive species generation in these anatomic sites. This experiment was conducted with the approval of the University of Pittsburgh IACUC (protocol # 1009934 A). Red, DMPO; Blue, DAPI; Green, actin; 2°, secondary antibody.