Novel Flurometric Tool to Assess Mitochondrial Redox State of Isolated Perfused Rat Lungs After Exposure to Hyperoxia

Abstract

Recently we demonstrated the utility of optical fluorometry to detect a change in the redox status of mitochondrial autofluorescent coenzymes NADH (Nicotinamide Adenine Dinucleotide) and FAD (oxidized form of Flavin Adenine Dinucleotide (FADH2,)) as a measure of mitochondrial function in isolated perfused rat lungs (IPL). The objective of this study was to utilize optical fluorometry to evaluate the effect of rat exposure to hyperoxia (>95% O2 for 48 hours) on lung tissue mitochondrial redox status of NADH and FAD in a nondestructive manner in IPL. Surface NADH and FAD signals were measured before and after lung perfusion with perfusate containing rotenone (ROT, complex I inhibitor), potassium cyanide (KCN, complex IV inhibitor), and/or pentachlorophenol (PCP, uncoupler). ROT- or KCN-induced increase in NADH signal is considered a measure of complex I activity, and KCN-induced decrease in FAD signal is considered a measure of complex II activity. The results show that hyperoxia decreased complex I and II activities by 63% and 55%, respectively, as compared to lungs of rats exposed to room air (normoxic rats). Mitochondrial complex I and II activities in lung homogenates were also lower (77% and 63%, respectively) for hyperoxic than for normoxic lungs. These results suggest that the mitochondrial matrix is more reduced in hyperoxic lungs than in normoxic lungs, and demonstrate the ability of optical fluorometry to detect a change in mitochondrial redox state of hyperoxic lungs prior to histological changes characteristic of hyperoxia.

Protection by Inhaled Hydrogen Therapy in a Rat Model of Acute Lung Injury can be Tracked in vivo Using Molecular Imaging

“…Table 7 shows that rat exposure to hyperoxia for 48 hrs increased FWHM time by ~50%. This suggests that ΔΨ m recovery from ADP-induced depolarization in mitochondria from hyperoxia lungs was substantially slower than that in normoxia lungs, consistent with decreases in complex I and II activities in lungs of hyperoxia rats (22). Table 7 shows that the ΔΨ m recovery time (FWHM) from ADP-stimulated depolarization, which increased with hyperoxia, partially reversed with hyperoxia+H 2 , consistent with the ability of H 2 to protect mitochondria from functional degradation by hyperoxia-induced oxidative stress.…”

“…We previously demonstrated significant decreases in complex I and II activities in lung of rats exposed to hyperoxia for 48 hrs (22). Mitochondrial DNA (mtDNA) is highly sensitive to reactive oxygen species (ROS) (29).…”

99MTc-Hexamethylpropyleneamine Oxime Imaging for Early Detection of Acute Lung Injury in Rats Exposed to Hyperoxia or Lipopolysaccharide Treatment

“…This component of the enhanced HMPAO uptake could be due to the increased capillary endothelial permeability as measured by pulmonary endothelial filtration coefficient ( K f ) (Table 3) or altered mitochondrial redox state. Previously, we demonstrated that rat exposure to > 95% O 2 for 48 hrs decreased lung tissue mitochondrial complex I and II activities and altered mitochondrial redox state (32). …”

Quantitative optical measurement of mitochondrial superoxide dynamics in pulmonary artery endothelial cells

“… 67 – 70 PCP dissipates the proton gradient by consuming the proton motive force via increased proton leak back into the matrix. A recent study reported that PCP oxidizes the ETC and increase the activity of complex I, 71 leading to the production of more protons and transfer of more electrons along the ETC. Lack of proton gradient for phosphorylation and polarized ΔΨm, activates a mechanism to compensate for the uncoupling effect of PCP, by increasing proton pumping and respiration, in an attempt to reestablish the proton gradient.…”