Date of Award

May 2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Engineering

First Advisor

Mahsa Ranji

Committee Members

Nader Sheibani, George Hanson, Jun Zhang, Ilya Avdeev

Keywords

Lung Tissue, NADH FAD, Optical Studies, Oxidative Stress, Rodent Models

Abstract

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's 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.

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