Optical Studies of Oxidative Stress in Lung Tissue: Rodent Models

Objectives: There currently exists a need for reliable measurements of tissue metabolic state at cellular levels. The objective of this research was to study tools capable of evaluating cellular redox states in intact tissue. To meet this goal, three different instruments (cryoimager, fluorometer, and fluorescent microscope) were used to study the metabolism and functions of the mitochondria at different levels and regimes (cryo, ex vivo, in vivo and in vitro). Introduction: Through optical monitoring of autofluorescent mitochondrial metabolic coenzymes, as well as exogenous fluorophores, the state of mitochondria can be probed in real time in many intact organs and in vitro. Autofluorescent mitochondrial metabolic coenzymes, studied here, include NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), and the ratio of these fluorophores, referred to as the mitochondrial redox ratio (RR), can be used as a quantitative metabolic marker of the tissue. Exogenous fluorophores include but are not limited to tetramethylrhodamine (TMRM) and Mito-SOX, which are used to evaluate the mitochondrial membrane potential and level of reactive oxygen species (ROS) in the mitochondria, respectively. Methods: Different optical imaging and acquisition techniques were studied to evaluate oxidative stress in lung tissue and cells in cryogenic temperatures, in vivo, ex vivo, and in vitro. Though in essence the underlying technological and biological principles appear to be the same, imaging in each of these regimes imposed unique challenges requiring significantly different approaches to system design, data acquisition, and processing. A brief description of each technique is provided here and each is described in detail in the following chapters. The first device utilized is a cryoimager, which sequentially slices tissue and acquires fluorescence images of up to five fluorophores in cryogenic temperatures (-40oC). Rapid freezing of organs preserves the tissue\u27s metabolic state and subsequent low temperature fluorescence imaging (cryoimaging) provides high fluorescence quantum yield as compared with room temperature. Sequential slicing of the tissue provides 3D spatial distribution of NADH and FAD fluorescence intensities throughout the tissue. These studies were conducted using the cryoimager in the Biophotonics Lab on different models of lung injuries including ischemia, hyperoxia, and BPD (bronchopulmonary dysplasia). The second device is a fluorometer, which was designed and implemented in the Biophotonics Lab. It is capable of monitoring the dynamics of the metabolism of the tissue through the use of optical surface fluorescence measurements of NADH and FAD. The ratio of these fluorophores, referred to as the mitochondrial redox ratio (RR), can be used as a quantitative metabolic marker of tissue. Surface fluorescence signals from NADH and FAD were acquired in the absence (baseline) and presence of metabolic perturbers (e.g. pentachlorophenol, rotenone, or potassium cyanide), in the presence of blood, and eventually in vivo. The third instrument, a fluorescent microscope, is used to image slides and dishes containing stained cells (e.g. endothelial cells, perycites, or fibroblasts) from lungs, hearts, and retinas to study their structure and dynamics at cellular level. Images of retinas were classified as normal or injured using developed cytometry tools and morphologic parameters. For heart and lung, the dynamics of concentration of reactive oxygen species (mainly superoxide) and calcium is monitored over time in cultured live cells. Results: In the cryogenic temperatures, lung treatment with KCN (inhibitor of Complex IV), resulted in an increase in RR and sets the upper limit of the NADH signal level while injured lungs (BPD model, hyperoxia and IR) showed a more oxidized chain compared with control lungs, and as a result more oxidative stress. In ex vivo fluorometric studies, an increase in RR from chain inhibitors (including KCN and rotenone), and a decrease in the same due to an uncoupler (PCP), all from baseline was observed which was consistent with the cryoimaging results. The same experiments in isolated perfused lungs previously treated with hyperoxia showed the same direction but different levels indicating the impairment in different complexes due to hyperoxia. Segmentation algorithm developed here showed 90% accuracy comparing to manual counting, and studying the cells in retina slides confirms apoptosis and oxidative stress in retinas from injured mice. In live cells, studying the dynamics of calcium concentration in the presence of different perturbations enabled us to study the behavior of mitochondrial regulated calcium channels. Also, changes in the Mito-SOX channel gave us the dynamics of mitochondrial ROS in the presence of chain perturbers (chemicals and gas). Conclusion: Optical instrumentation combined with signal and image processing tools provide quantitative physiological and structural information of diseased tissue due to oxidative stress

Surface Fluorescence Studies of Tissue Mitochondrial Redox State in Isolated Perfused Rat Lungs

We designed a fiber-optic-based optoelectronic fluorometer to measure emitted fluorescence from the auto-fluorescent electron carriers NADH and FAD of the mitochondrial electron transport chain (ETC). The ratio of NADH to FAD is called the redox ratio (RR = NADH/FAD) and is an indicator of the oxidoreductive state of tissue. We evaluated the fluorometer by measuring the fluorescence intensities of NADH and FAD at the surface of isolated, perfused rat lungs. Alterations of lung mitochondrial metabolic state were achieved by the addition of rotenone (complex I inhibitor), potassium cyanide (KCN, complex IV inhibitor) and/or pentachlorophenol (PCP, uncoupler) into the perfusate recirculating through the lung. Rotenone- or KCN-containing perfusate increased RR by 21 and 30%, respectively. In contrast, PCP-containing perfusate decreased RR by 27%. These changes are consistent with the established effects of rotenone, KCN, and PCP on the redox status of the ETC. Addition of blood to perfusate quenched NADH and FAD signal, but had no effect on RR. This study demonstrates the capacity of fluorometry to detect a change in mitochondrial redox state in isolated perfused lungs, and suggests the potential of fluorometry for use in in vivo experiments to extract a sensitive measure of lung tissue health in real-time

Optical Imaging of Lipopolysaccharide-induced Oxidative Stress in Acute Lung Injury from Hyperoxia and Sepsis

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 mitochondrial metabolic coenzymes, NADH and FAD in four different groups of rats. The ratio of these fluorescence signals (NADH/FAD), referred to as NADH redox ratio (NADH RR) has been used as an indicator of tissue metabolism in injuries. Here, we investigated whether the changes in metabolic state can be used as a marker of oxidative stress caused by hyperoxia and bacterial lipopolysaccharide (LPS) exposure in neonatal rat lungs. We examined the tissue redox states of lungs from four groups of rat pups: normoxic (21% O2) pups, hyperoxic (90% O2) pups, pups treated with LPS (normoxic + LPS), and pups treated with LPS and hyperoxia (hyperoxic + LPS). Our results show that hyperoxia oxidized the respiratory chain as reflected by a ~ 31% decrease in lung tissue NADH RR as compared to that for normoxic lungs. LPS treatment alone or with hyperoxia had no significant effect on lung tissue NADH RR as compared to that for normoxic or hyperoxic lungs, respectively. Thus, NADH RR serves as a quantitative marker of oxidative stress level in lung injury caused by two clinically important conditions: hyperoxia and LPS exposure

Burn and Hypertension: How Are They Related?

Background: A burn tissue injury is one of the most severe forms of trauma which results in severe life-threatening disturbances. Burn injury has many morbid complications, so it needs a multi-disciplinary care team according to the burn center to reduce its mortality and morbidity. Methods: This article aims to review drawbacks and complications associated with the burning injury including Acute Kidney Injury (AKI), Acute lung injury, Heart Failure, Electrolyte imbalance, intra-abdominal hypertension in children and adult burn patients, and recent challenging treatments. Results: Improved understanding of the pathophysiology of burn-induced complications can contribute to organizing a well-treatment plan, which leads to improved outcomes. Conclusions: Herein, the evidence available on the management of all burn induced-complications is summarized

Data associated with Retinal oxidative stress at the onset of diabetes determined by synchrotron FTIR widefield imaging: towards diabetes pathogenesis and Temporal diabetes-induced biochemical changes in distinctive layers of mouse retina

This data folder is associated with the following publications in the Analyst journal and the Scientific Reports: 1. Retinal oxidative stress at the onset of diabetes determined by synchrotron FTIR widefield imaging: towards diabetes pathogenesis. (E. Aboualizadeh, et al. February 2017) DOI: 10.1039/C6AN02603F Citation: Aboualizadeh, E., Ranji, M., Sorenson, C. M., Sepehr, R., Sheibani, N., & Hirschmugl, C. J. (2017). Retinal oxidative stress at the onset of diabetes determined by synchrotron FTIR widefield imaging: towards diabetes pathogenesis. Analyst, 142(7), 1061-1072. AND 2. Temporal diabetes-induced biochemical changes in distinctive layers of mouse retina. (E. Aboualizadeh, et al. January 2018) DOI: 10.1038/s41598-018-19425-8 Citation: Aboualizadeh, E., Sorenson, C. M., Schofield, A. J., Unger, M., Sheibani, N., & Hirschmugl, C. J. (2018). Temporal diabetes-induced biochemical changes in distinctive layers of mouse retina. Scientific reports, 8(1), 1096. Abstract of paper 1: Diabetic retinopathy is a microvascular complication of diabetes that can lead to blindness. In the present study, we aimed to determine the nature of diabetes-induced, highly localized biochemical changes in the neuroretina at the onset of diabetes. High-resolution synchrotron Fourier transform infrared (s-FTIR) wide field microscopy coupled with multivariate analysis (PCA–LDA) was employed to identify biomarkers of diabetic retinopathy with spatial resolution at the cellular level. We compared the retinal tissue prepared from 6-week-old Ins2Akita/+ heterozygous (Akita/+, N = 6; a model of diabetes) male mice with the wild-type (control, N = 6) mice. Male Akita/+ mice become diabetic at 4-weeks of age. Significant differences (P \u3c 0.001) in the presence of biomarkers associated with diabetes and segregation of spectra were achieved. Differentiating IR bands attributed to nucleic acids (964, 1051, 1087, 1226 and 1710 cm−1), proteins (1662 and 1608 cm−1) and fatty acids (2854, 2923, 2956 and 3012 cm−1) were observed between the Akita/+ and the WT samples. A comparison between distinctive layers of the retina, namely the photoreceptor retinal layer (PRL), outer plexiform layer (OPL), inner nucleus layer (INL) and inner plexiform layer (IPL) suggested that the photoreceptor layer is the most susceptible layer to oxidative stress in short-term diabetes. Spatially-resolved chemical images indicated heterogeneities and oxidative-stress induced alterations in the diabetic retina tissue morphology compared with the WT retina. In this study, the spectral biomarkers and the spatial biochemical alterations in the diabetic retina and in specific layers were identified for the first time. We believe that the conclusions drawn from these studies will help to bridge the gap in our understanding of the molecular and cellular mechanisms that contribute to the pathobiology of diabetic retinopathy. Abstract of paper 2: To discover the mechanisms underlying the progression of diabetic retinopathy (DR), a more comprehensive understanding of the biomolecular processes in individual retinal cells subjected to hyperglycemia is required. Despite extensive studies, the changes in the biochemistry of retinal layers during the development of DR are not well known. In this study, we aimed to determine a more detailed understanding of the natural history of DR in Akita/+ (type 1 diabetes model) male mice with different duration of diabetes. Employing label-free spatially resolved Fourier transform infrared (FT-IR) chemical imaging engaged with multivariate analysis enabled us to identify temporal-dependent reproducible biomarkers of the individual retinal layers from mice with 6 weeks,12 weeks, 6 months, and 10 months of age. We report, for the first time, the nature of the biochemical alterations over time in the biochemistry of distinctive retinal layers namely photoreceptor retinal layer (PRL), inner nuclear layer (INL), and plexiform layers (OPL, IPL). Moreover, we present the molecular factors associated with the changes in the protein structure and cellular lipids of retinal layers induced by different duration of diabetes. Our paradigm provides a new conceptual framework for a better understanding of the temporal cellular changes underlying the progression of DR. The data folder contains all the data that has been included in this publication. The data files are stored as OPUS files and DPT files. There are files based on the age of mice ranging from 6-weeks to 10 months old diabetic and wild-type mice. The bright field images are also stored in the data folder. The tables contain FT-IR spectra from diabetic and control retina at different duration of diabetes, where each column is one spectrum and the first column in each table shows the wavenumber range. The processed data in this folder are the processed OPUS data in Irydis software and they are saved as PXP file. We included all these data in these 2 mentioned publications. Please feel free to contact either Dr. Carol Hirschmugl ([email protected]) or Dr. Ebrahim Aboualizadeh ([email protected]) for any questions or concerns regarding this data. This second article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder

Anti-Cancer Effect of Melatonin via Downregulation of Delta-Like Ligand 4 in Estrogen-Responsive Breast Cancer Cells

Background: The Notch signaling pathway has a key role in angiogenesis and Delta-Like Ligand 4 (DLL4) is one of the main ligands of Notch involved in cell proliferation in sprouting vessels. Objective: In this study, we aimed to evaluate the expression of DLL4 in primary breast tumors and to examine the effect of melatonin on DLL4 expression in vitro. Methods: Eighty-five breast tumor and paired adjacent non-tumor tissue samples were collected. Apoptosis assay was performed on breast cancer cells to evaluate melatonin effects. Western blot and quantitative RT-PCR were used to measure DLL4 expression. Then, we investigated the effect of melatonin on the expression of DLL4 in four breast cancer cell lines at RNA and protein levels. We also performed a probabilistic neural network analysis to study genes closely associated with DLL4 expression. Results: Our results showed a significantly higher expression of DLL4 in tumor tissues compared to non-tumor tissues (P = 0.027). Melatonin treatment substantially attenuated DLL4 expression in BT474 and MCF-7 cells, but not in SK-BR-3 and MDA-MB-231 cells. Also, melatonin induced apoptosis in all four cell lines. Network analysis revealed a set of 15 genes that had close association and interaction with DLL4. DLL4 was overexpressed in breast cancer tissues as compared to the non-tumor tissues. Conclusion: It can be concluded that melatonin treatment attenuated DLL4 expression only in estrogen-responsive breast cancer cells and is able to induce apoptosis in breast cancer cells