Biomedical Engineering and Bioengineering Commons^™

Detection Of Hydrogen Peroxide Production In The Isolated Rat Lung Using Amplex Red, Said H. Audi, Nina Friedly, Ranjan K. Dash, Andreas M. Beyer, Anne V. Clough, Elizabeth R. Jacobs

Biomedical Engineering Faculty Research and Publications

The objectives of this study were to develop a robust protocol to measure the rate of hydrogen peroxide (H₂O₂) production in isolated perfused rat lungs, as an index of oxidative stress, and to determine the cellular sources of the measured H₂O₂ using the extracellular probe Amplex red (AR). AR was added to the recirculating perfusate in an isolated perfused rat lung. AR’s highly fluorescent oxidation product resorufin was measured in the perfusate. Experiments were carried out without and with rotenone (complex I inhibitor), thenoyltrifluoroacetone (complex II inhibitor), antimycin A (complex III inhibitor), potassium …

Hyperoxia Causes Mitochondrial Fragmentation In Pulmonary Endothelial Cells By Increasing Expression Of Pro-Fission Proteins, Cui Ma, Andreas M. Beyer, Matthew J. Durand, Anne V. Clough, Daling Zhu, Laura Norwood Toro, Maia Terashvili, Johnathan D. Ebben, R. Blake Hill, Said H. Audi, Meetha Medhora, Elizabeth R. Jacobs

Biomedical Engineering Faculty Research and Publications

Objective—We explored mechanisms that alter mitochondrial structure and function in pulmonary endothelial cells (PEC) function after hyperoxia.

Approach and Results—Mitochondrial structures of PECs exposed to hyperoxia or normoxia were visualized and mitochondrial fragmentation quantified. Expression of pro-fission or fusion proteins or autophagy-related proteins were assessed by Western blot. Mitochondrial oxidative state was determined using mito-roGFP. Tetramethylrhodamine methyl ester estimated mitochondrial polarization in treatment groups. The role of mitochondrially derived reactive oxygen species in mt-fragmentation was investigated with mito-TEMPOL and mitochondrial DNA (mtDNA) damage studied by using ENDO III (mt-tat-endonuclease III), a protein that repairs mDNA damage. Drp-1 (dynamin-related …

99mtc-Hexamethylpropyleneamine Oxime Imaging For Early Detection Of Acute Lung Injury In Rats Exposed To Hyperoxia Or Lipopolysaccharide Treatment, Said H. Audi, Anne V. Clough, Steven T. Haworth, Meetha Medhora, Mahsa Ranji, John C. Densmore, Elizabeth R. Jacobs

Biomedical Engineering Faculty Research and Publications

^99mTc-Hexamethylpropyleneamine oxime (HMPAO) is a clinical single-photon emission computed tomography biomarker of tissue oxidoreductive state. Our objective was to investigate whether HMPAO lung uptake can serve as a preclinical marker of lung injury in two well-established rat models of human acute lung injury (ALI).

Rats were exposed to >95% O₂ (hyperoxia) or treated with intratracheal lipopolysaccharide (LPS), with first endpoints obtained 24 h later. HMPAO was administered intravenously before and after treatment with the glutathione-depleting agent diethyl maleate (DEM), scintigraphy images were acquired, and HMPAO lung uptake was quantified from the images. We also measured breathing rates, heart …

Optical Imaging Of Lipopolysaccharide-Induced Oxidative Stress In Acute Lung Injury From Hyperoxia And Sepsis, Reyhaneh Sepehr, Said H. Audi, Sepideh Maleki, Kevin Staniszewski, Annie L. Eis, Girija G. Konduri, Mahsa Ranji

Biomedical Engineering Faculty Research and Publications

Reactive oxygen species (ROS) have been implicated in the pathogenesis of many acute and chronic pulmonary disorders such as acute lung injury (ALI) in adults and bronchopulmonary dysplasia (BPD) in premature infants. Bacterial infection and oxygen toxicity, which result in pulmonary vascular endothelial injury, contribute to impaired vascular growth and alveolar simplification seen in the lungs of premature infants with BPD. Hyperoxia induces ALI, reduces cell proliferation, causes DNA damage and promotes cell death by causing mitochondrial dysfunction. The objective of this study was to use an optical imaging technique to evaluate the variations in fluorescence intensities of the auto-fluorescent …

Quantifying Mitochondrial And Plasma Membrane Potentials In Intact Pulmonary Arterial Endothelial Cells Based On Extracellular Disposition Of Rhodamine Dyes, Zhuohui Gan, Said H. Audi, Robert D. Bongard, Kathryn M. Gauthier, Marilyn P. Merker

Biomedical Engineering Faculty Research and Publications

Our goal was to quantify mitochondrial and plasma potential (Δψ_m and Δψ_p) based on the disposition of rhodamine 123 (R123) or tetramethylrhodamine ethyl ester (TMRE) in the medium surrounding pulmonary endothelial cells. Dyes were added to the medium, and their concentrations in extracellular medium ([R_e]) were measured over time. R123 [R_e] fell from 10 nM to 6.6 ± 0.1 (SE) nM over 120 min. TMRE [R_e] fell from 20 nM to a steady state of 4.9 ± 0.4 nM after ∼30 min. Protonophore or high K⁺ concentration ([K⁺]), …

Distribution Of Capillary Transit Times In Isolated Lungs Of Oxygen-Tolerant Rats, Madhavi Ramakrishna, Zhuohui Gan, Anne V. Clough, Robert C. Molthen, David L. Roerig, Said H. Audi

Biomedical Engineering Faculty Research and Publications

Rats pre-exposed to 85% O₂ for 5–7 days tolerate the otherwise lethal effects of 100% O₂. The objective was to evaluate the effect of rat exposure to 85% O₂ for 7 days on lung capillary mean transit time (t¯c) and distribution of capillary transit times (h _c(t)). This information is important for subsequent evaluation of the effect of this hyperoxia model on the redox metabolic functions of the pulmonary capillary endothelium. The venous concentration vs. time outflow curves of fluorescein isothiocyanate labeled dextran (FITC-dex), an intravascular indicator, and coenzyme Q₁ hydroquinone …