Date of Award

Spring 2018

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biomedical Engineering

First Advisor

Audi, Said

Second Advisor

Clough, Anne

Third Advisor

Dash, Ranjan

Abstract

Oxidative stress, the imbalance between production of oxidants or reactive oxygen species (ROS) and antioxidant activity, plays a key role in the pathogenesis of acute and chronic lung diseases. The objective of this thesis was to develop a robust protocol for measuring the rate of H2O2 production in isolated perfused rat lungs and to determine the cellular sources of that rate using Amplex Red (AR). For a given lung, AR (25 mM) along with horseradish peroxidase (5 U/ml) and ascorbate oxidase (1U/ml) were added to a perfusate reservoir that was recirculated through the lungs and sampled at 5 minute intervals to measure the emission signal (454 nm/610 nm). Experiments were carried without and with the inhibitors rotenone (40 M), thenoyltrifluoroacetone (20 M), antimycin A (3.76 M), potassium cyanide (2 mM), or diohenylene iodonium (5M) added to the recirculating perfusate. In addition, we evaluated the effect of %O2 ventilation on H2O2 production. For lungs from control rats, the results show that inhibiting mitochondrial complex II reduced this rate by 76 ± 3%, and inhibiting NOX reduced it by another 23 ± 2%. The results also show that inhibiting complex I had a small (13% ± 4%), but significant effect on the rate, whereas inhibiting complex III had no significant effect on this rate. Inhibition of complex IV increased the rate or ROS production by 310% ± 43%. Furthermore, the results show that increasing % O2 in the ventilation gas mixture from 15% to 95% O2 had a relatively small (27± 3 %), but significant effect on this rate, and that this O2-dependent increase was mostly non-mitochondrial. The results of this study suggest complex II as a potentially important source of ROS and a potential target for mitigating oxidative stress, and that most of the hyperoxia-enhanced lung rate H2O2 release is from NAD(P)H oxidase rather than mitochondrial sources. To the best of our knowledge, this is the first study measuring the rate of H2O2 release from isolated perfused rat lungs, identifying the main sources of this rate under physiological conditions, and evaluating the effect of acute hyperoxia on this rate.

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