Reactive oxygen species (ROS) play an essential role in facilitating signal transduction processes within the cell and modulating the injuries. However, the generation of ROS is tightly controlled both spatially and temporally within the cell, making the study of ROS dynamics particularly difficult. This study present a novel protocol to quantify the dynamic of the mitochondrial superoxide as a precursor of reactive oxygen species. To regulate the mitochondrial superoxide level, metabolic perturbation was induced by administration of potassium cyanide (KCN). The presented method was able to monitor and measure the superoxide production rate over time. Our results demonstrated that the metabolic inhibitor, potassium cyanide (KCN) induced a significant increase in the rate of superoxide production in mitochondria of fetal pulmonary artery endothelial cells (FPAEC). Presented method sets the stage to study different ROS mediated injuries in vitro.
Previously we demonstrated the utility of optical fluorometry to evaluate lung tissue mitochondrial redox state
in isolated perfused rats lungs under various chemically-induced respiratory states. The objective of this study
was to evaluate the effect of acute ischemia on lung tissue mitochondrial redox state in vivo using optical
fluorometry. Under ischemic conditions, insufficient oxygen supply to the mitochondrial chain should reduce
the mitochondrial redox state calculated from the ratio of the auto-fluorescent mitochondrial metabolic
coenzymes NADH (Nicotinamide Adenine Dinucleotide) and FAD (Flavoprotein Adenine Dinucleotide). The
chest of anesthetized, and mechanically ventilated Sprague-Dawley rat was opened to induce acute ischemia by
clamping the left hilum to block both blood flow and ventilation to one lung for approximately 10 minutes.
NADH and FAD fluorescent signals were recorded continuously in a dark room via a fluorometer probe placed
on the pleural surface of the left lung. Acute ischemia caused a decrease in FAD and an increase in NADH,
which resulted in an increase in the mitochondrial redox ratio (RR=NADH/FAD). Restoration of blood flow
and ventilation by unclamping the left hilum returned the RR back to its baseline. These results (increase in RR
under ischemia) show promise for the fluorometer to be used in a clinical setting for evaluating the effect of
pulmonary ischemia-reperfusion on lung tissue mitochondrial redox state in real time.
Oxidative stress (OS), which increases during retinal degenerative disorders, contributes to photoreceptor cell loss. The
objective of this study was to investigate the changes in the metabolic state of the eye tissue in rodent models of retinitis pigmentosa by using the cryofluorescence imaging technique. The mitochondrial metabolic coenzymes NADH and FADH<sub>2</sub> are autofluorescent and can be monitored without exogenous labels using optical techniques. The NADH redox ratio (RR), which is the ratio of the fluorescence intensity of these fluorophores (NADH/FAD), was used as a quantitative diagnostic marker. The NADH RR was examined in an established rodent model of retinitis pigmentosa (RP), the P23H rat, and compared to that of control Sprague-Dawley (SD) rats and P23H NIR treated rats. Our results demonstrated 24% decrease in the mean NADH RR of the eyes from P23H transgenic rats compared to normal rats and 20% increase in the mean NADH RR of the eyes from the P23H NIR treated rats compared to P23H non-treated rats.
Oxidative stress (OS) and mitochondrial dysfunction contribute to photoreceptor cell loss in retinal degenerative disorders. The metabolic state of the retina in a rodent model of retinitis pigmentosa (RP) was investigated using a cryo-fluorescence imaging technique. The mitochondrial metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent and can be monitored without exogenous labels using optical techniques. The cryo-fluorescence redox imaging technique provides a quantitative assessment of the metabolism. More specifically, the ratio of the fluorescence intensity of these fluorophores (NADH/FAD), the NADH redox ratio (RR), is a marker of the metabolic state of the tissue. The NADH RR and retinal function were examined in an established rodent model of RP, the P23H rat compared to that of nondystrophic Sprague-Dawley (SD) rats. The NADH RR mean values were 1.11±0.03 in the SD normal and 0.841±0.01 in the P23H retina, indicating increased OS in the P23H retina. Electroretinographic data revealed a significant reduction in photoreceptor function in P23H animals compared to SD nozrmal rats. Thus, cryo-fluorescence redox imaging was used as a quantitative marker of OS in eyes from transgenic rats and demonstrated that alterations in the oxidative state of eyes occur during the early stages of RP.
Ventilation with enhanced fractions of O2 (hyperoxia) is a common and necessary treatment for hypoxemia in patients with lung failure, but prolonged exposure to hyperoxia causes lung injury. Ischemia-reperfusion (IR) injury of lung tissue is common in lung transplant or crush injury to the chest. These conditions are associated with apoptosis and decreased survival of lung tissue. The objective of this work is to use cryoimaging to evaluate the effect of exposure to hyperoxia and IR injury on lung tissue mitochondrial redox state in rats. The autofluorescent mitochondrial metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are electron carriers in ATP generation. These intrinsic fluorophores were imaged for rat lungs using low-temperature fluorescence imaging (cryoimaging). Perfused lungs from four groups of rats were studied: normoxia (control), control perfused with an mitochondrial complex IV inhibitor (potassium cyanide, KCN), rats exposed to hyperoxia (85% O2) for seven days, and from rats subjected to lung IR in vivo 24 hours prior to study. Each lung was sectioned sequentially in the transverse direction, and the images were used to reconstruct a three-dimensional (3-D) rendering. In KCN perfused lungs the respiratory chain was more reduced, whereas hyperoxic and IR lung tissue have a more oxidized respiratory chain than control lung tissue, consistent with previously measured mitochondrial dysfunction in both hyperoxic and IR lungs.
Oxidative stress (OS), which increases during diabetes, exacerbates the development and progression of diabetes
complications including renal vascular and proximal tubule cell dysfunction. The objective of this study was to
investigate the changes in the metabolic state of the tissue in diabetic mice kidneys using fluorescence imaging.
Mitochondrial metabolic coenzymes NADH (Nicotinamide Adenine Dinucleotide), and FADH-2 (Flavin Adenine
Dinucleotide) are autofluorescent and can be monitored without exogenous labels by optical techniques. The ratio of the
fluorescence intensity of these fluorophores, (NADH/FAD), called the NADH redox ratio (RR), is a marker of metabolic
state of a tissue. We examined mitochondrial redox states of kidneys from diabetic mice, Akita/+ and its control wild
type (WT) for a group of 8- and 12-week-old mice. Average intensity and histogram of maximum projected images of
FAD, NADH, and NADH RR were calculated for each kidney. Our results indicated a 17% decrease in the mean NADH
RR of the kidney from 8-week-old mice compared with WT mice and, a 30% decrease in the mean NADH RR of kidney
from12-week-old mice compared with WT mice. These results indicated an increase in OS in diabetic animals and its
progression over time. Thus, NADH RR can be used as a hallmark of OS in diabetic kidney allowing temporal
identification of oxidative state.
Through the monitoring of the auto-fluorescent mitochondrial metabolic coenzymes, NADH (Nicotinamide Adenine
Dinucleotide) and FAD (Flavoprotein Adenine Dinucleotide), the redox state of metabolism can be probed in real time in
many intact organs, but its use has not been fully developed in lungs. The ratio of these fluorophores, (NADH/FAD),
referred to as the mitochondrial redox ratio (RR), can be used as a quantitative metabolic marker of tissue. We have
designed a fluorometer that can be used to monitor lung surface NADH and FAD fluorescence in isolated perfused
lungs. Surface fluorescence NADH and FAD signals were acquired in the absence (control) and presence of
pentachlorophenol (PCP), rotenone, and potassium cyanide (KCN). Rotenone, an inhibitor of complex I, increased RR
by 18%, predominantly due to an increase in NADH signal. KCN, an inhibitor of complex IV reduced the chain and
resulted in an increase of 33% in RR, as a result of 23% increase in NADH and 8% in FAD . PCP, an uncoupler which
oxidizes the respiratory chain, decreased RR by 18% as a result of 14% decrease in NADH signal and 4% increase in
FAD signal. These results demonstrate the ability of surface fluorometry to detect changes in lung tissue mitochondrial
redox state in isolated perfused lungs.
Retinopathic injuries are a common symptom of many diseases. However, if detected early, much of the damage caused
by these injuries can be prevented, or in some cases reversed. In this study, images of retinas were classified as normal or
injured using the vascular cell count, vasculature coverage, and vessel caliber. To model retinal vasculopathies, retinal
vasculature from mice with the BCL-2 gene either partially or completely knocked out were compared. The bcl-2 gene is
a critical regulator of apoptosis and angiogenesis, and therefore its absence has a significant impact on the number of
vascular cells and vasculature complexity. When the aforementioned features were extracted from the images,
classification was performed using a majority vote between a linear classifier, k-nearest-neighbors classification, and a
support vector machine. This resulted in a classification accuracy of 81% using the "leave one out" error determination
The objective of this study was to demonstrate the utility of optical cryoimaging and fluorometry to evaluate tissue redox
state of the mitochondrial metabolic coenzymes NADH (Nicotinamide Adenine Dinucleotide) and FAD (Flavin Adenine
Dinucleotide) in intact rat lungs. The ratio (NADH/FAD), referred to as mitochondrial redox ratio (RR), is a measure of
the lung tissue mitochondrial redox state. Isolated rat lungs were connected to a ventilation-perfused system. Surface
NADH and FAD fluorescence signals were acquired before and after lung perfusion in the absence (control perfusate) or
presence of potassium cyanide (KCN, complex IV inhibitor) to reduce the mitochondrial respiratory chain (state 5
respiration). Another group of lungs were perfused with control perfusate or KCN-containing perfusate as above, after
which the lungs were deflated and frozen rapidly for subsequent 3D cryoimaging. Results demonstrate that lung
treatment with KCN increased lung surface NADH signal by 22%, decreased FAD signal by 8%, and as result increased
RR by 31% as compared to control perfusate (baseline) values. Cryoimaging results also show that KCN increased mean
lung tissue NADH signal by 37%, decreased mean FAD signal by 4%, and increased mean RR by 47%. These results
demonstrate the utility of these optical techniques to evaluate the effect of pulmonary oxidative stress on tissue
mitochondrial redox state in intact lungs.