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

Reactive oxygen species (ROS) play a vital role in cell signaling and redox regulation, but when present in excess, lead to numerous pathologies. Detailed quantitative characterization of mitochondrial superoxide anion ( O2•- ) production in fetal pulmonary artery endothelia cells (PAECs) has never been reported. The aim of this study is to assess mitochondrial O2•- production in cultured PAECs over time using a novel quantitative optical approach. The rate, the sources, and the dynamics of O2•- production were assessed using targeted metabolic modulators of the mitochondrial electron transport chain (ETC) complexes, specifically an uncoupler and inhibitors of the various ETC complexes, and inhibitors of extra-mitochondrial sources of O2•- . After stabilization, the cells were loaded with nanomolar mitochondrial-targeted hydroethidine (Mito-HE, MitoSOX) online during the experiment without washout of the residual dye. Time-lapse fluorescence microscopy was used to monitor the dynamic changes in O2•- fluorescence intensity over time in PAECs. The transient behaviors of the fluorescence time course showed exponential increases in the rate of O2•- production in the presence of the ETC uncoupler or inhibitors. The most dramatic and the fastest increase in O2•- production was observed when the cells were treated with the uncoupling agent, PCP. We also showed that only the complex IV inhibitor, KCN, attenuated the marked surge in O2•- production induced by PCP. The results showed that mitochondrial respiratory complexes I, III and IV are sources of O2•- production in PAECs, and a new observation that ROS production during uncoupling of mitochondrial respiration is mediated in part via complex IV. This novel method can be applied in other studies that examine ROS production under stress condition and during ROS-mediated injuries in vitro.

Time-lapse microscopy of oxidative stress demonstrates metabolic sensitivity of retinal pericytes under high glucose condition.

Hyperglycemia affects retinal vascular cell function, promotes the development and progression of diabetic retinopathy and ultimately causes vision loss. Oxidative stress, reactive oxygen species (ROS) in excess, is a key biomarker for diabetic retinopathy. Using time-lapse fluorescence microscopy, ROS dynamics was monitored and the metabolic resistivity of retinal endothelial cells (REC) and pericytes (RPC) was compared under metabolic stress conditions including high glucose (HG). In the presence of a mitochondrial stressor, REC exhibited a significant increase in the rate of ROS production compared with RPC. Thus, under normal glucose (NG), REC may utilize oxidative metabolism as the bioenergetic source, while RPC metabolic activity is independent of mitochondrial respiration. In HG condition, the rate of ROS production in RPC was significantly higher, whereas this rate remained unchanged in REC. Thus, under HG condition RPC may preferentially utilize oxidative metabolism, which results in increased rate of ROS production. In contrast, REC use glycolysis as their major bioenergetic source for ATP production, and consequently HG minimally affects their ROS levels. These observations are consistent with our previous studies where we showed HG condition has minimal effect on apoptosis of REC, but results in increased rate of apoptosis in RPC. Collectively, our results suggest that REC and RPC exhibit different metabolic activity preferences under different glucose conditions. Thus, protection of RPC from oxidative stress may provide an early point of intervention in development and progression of diabetic retinopathy.

Computational insertion of microcalcification clusters on mammograms: reader differentiation from native clusters and computer-aided detection comparison.

Mammographic computer-aided detection (CADe) devices are typically first developed and assessed for a specific "original" acquisition system. When developers are ready to apply their CADe device to a mammographic acquisition system, they typically assess the device with images acquired using the system. Collecting large repositories of clinical images containing verified lesion locations acquired by a system is costly and time consuming. We previously developed an image blending technique that allows users to seamlessly insert regions of interest (ROIs) from one medical image into another image. Our goal is to assess the performance of this technique for inserting microcalcification clusters from one mammogram into another, with the idea that when fully developed, our technique may be useful for reducing the clinical data burden in the assessment of a CADe device for use with an image acquisition system. We first perform a reader study to assess whether experienced observers can distinguish between computationally inserted and native clusters. For this purpose, we apply our insertion technique to 55 clinical cases. ROIs containing microcalcification clusters from one breast of a patient are inserted into the contralateral breast of the same patient. The analysis of the reader ratings using receiver operating characteristic (ROC) methodology indicates that inserted clusters cannot be reliably distinguished from native clusters (area under the ROC curve = 0.58 ± 0.04 ). Furthermore, CADe sensitivity is evaluated on mammograms of 68 clinical cases with native and inserted microcalcification clusters using a commercial CADe system. The average by-case sensitivities for native and inserted clusters are equal, 85.3% (58/68). The average by-image sensitivities for native and inserted clusters are 72.3% and 67.6%, respectively, with a difference of 4.7% and a 95% confidence interval of [ - 2.1 11.6]. These results demonstrate the potential for using the inserted microcalcification clusters for assessing mammographic CADe devices.

Quantitative Assessment of Retinopathy Using Multi-parameter Image Analysis.

A multi-parameter quantification method was implemented to quantify retinal vascular injuries in microscopic images of clinically relevant eye diseases. This method was applied to wholemount retinal trypsin digest images of diabetic Akita/+, and bcl-2 knocked out mice models. Five unique features of retinal vasculature were extracted to monitor early structural changes and retinopathy, as well as quantifying the disease progression. Our approach was validated through simulations of retinal images. Results showed fewer number of cells (P = 5.1205e-05), greater population ratios of endothelial cells to pericytes (PCs) (P = 5.1772e-04; an indicator of PC loss), higher fractal dimension (P = 8.2202e-05), smaller vessel coverage (P = 1.4214e-05), and greater number of acellular capillaries (P = 7.0414e-04) for diabetic retina as compared to normal retina. Quantification using the present method would be helpful in evaluating physiological and pathological retinopathy in a high-throughput and reproducible manner.

Organ specific optical imaging of mitochondrial redox state in a rodent model of hereditary hemorrhagic telangiectasia-1.

Hereditary Hemorrhagic Telangiectasia-1 (HHT-1) is a vascular disease caused by mutations in the endoglin (Eng)/CD105 gene. The objective of this study was to quantify the oxidative state of a rodent model of HHT-1 using an optical imaging technique. We used a cryofluorescence imaging instrument to quantitatively assess tissue metabolism in this model. Mitochondrial redox ratio (FAD/NADH), FAD RR, was used as a quantitative marker of the metabolic status and was examined in the kidneys, and eyes of wild-type and Eng +/- mice. Kidneys and eyes from wild-type P21, 6W, and 10M old mice showed, respectively, a 9% (±2), 24% (±0.4), 15% (±1), and 23% (±4), 33% (±0.6), and 30% (±2) change in the mean FAD RR compared to Eng +/- mice at the same age. Thus, endoglin haploinsufficiency is associated with less oxidative stress in various organs and mitigation of angiogenesis.

Optical imaging of mitochondrial redox state in rodent models with 3-iodothyronamine.

This study used an optical technique to measure the effects of treating low (10 mg/kg) and high (25 mg/kg) doses of 3-iodothyronamine (T1AM) on the metabolism in the kidney and heart of mice. The ratio of two intrinsic fluorophores in tissue, (NADH/FAD), called the NADH redox ratio (NADH RR), is a marker of the metabolic state of the tissue. A cryofluorescence imaging instrument was used to provide a quantitative assessment of NADH RR in both kidneys and hearts in mice treated with 3-iodothyronamine. We compared those results to corresponding tissues in control mice. In the kidneys of mice treated with a high dose T1AM, the mean values of the maximum projection of NADH RR were 2.6 ± 0.6 compared to 3.20 ± 0.03 in control mice, indicating a 19% (± 0.4) significant increase in oxidative stress (OS) in the high dose-treated kidneys (P = 0.047). However, kidneys treated with a low dose of T1AM showed no difference in NADH RR compared to the kidneys of control mice. Furthermore, low versus high dose treatment of T1AM showed different responses in the heart than in the kidneys. The mean value of the maximum projection of NADH RR in the heart changed from 3.0 ± 0.3 to 3.2 ± 0.6 for the low dose and the high dose T1AM-treated mice, respectively, as compared to 2.8 ± 0.7 in control mice. These values correspond to a 9% (±0.5) (P = 0.045) and 14% (±0.5) (P = 0.008) significant increase in NADH RR in the T1AM-treated hearts, indicating that the high dose T1AM-treated tissues have reduced OS compared to the low dose-treated tissues or the control tissues. These results suggest that while T1AM at a high dose increases oxidative response in kidneys, it has a protective effect in the heart and may exert its effect through alternative pathways at different doses and at tissue specific levels.

Optical imaging of mitochondrial redox state in rodent model of retinitis pigmentosa.

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.