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 FADH2 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.
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.
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