Ross D. Shonat

Oxygen tension imaging in the mouse retina.

AbstractA newly developed microscope-based imaging system was used to measure the oxygen tension (PO2) inside the retinal and choroidal vessels of mice and to generate in vivo maps of retinal PO2. These maps were generated from the phosphorescence lifetimes of an injected palladium–porphyrin compound using a frequency-domain measurement. The system was fully calibrated and used to produce retinal PO2 maps at different inspiratory oxygen fractions. PO2 rose accordingly and predictably as inspiratory O2 was stepped from hypoxic to hyperoxic conditions. Important experimental and acquisition parameters necessary for applying phosphorescence lifetime imaging to the mouse eye were investigated, including camera exposure and intensifier gain settings. Because of a need to limit light exposure to the retina, PO2 map quality as measured by the coefficient of determination was investigated as a function of signal-to-noise and accumulated excitation energy deposition. With the development of this technology for use in mice, the potential for investigating the oxygen dynamics in genetically engineered mouse models of retinal disease, including diabetic retinopathy, glaucoma, and age-related macular degeneration, is advanced.

Critical PO2 of skeletal muscle in vivo

The main purpose of this study was to determine the interstitial oxygen tension at which aerobic metabolism becomes limited (critical PO(2)) in vivo in resting skeletal muscle. Using an intravital microscope system, we determined the interstitial oxygen tension at 20-micrometer-diameter tissue sites in rat spinotrapezius muscle from the phosphorescence lifetime decay of a metalloporphyrin probe during a 1-min stoppage of muscle blood flow. In paired experiments NADH fluorescence was measured at the same sites during flow stoppage. NADH fluorescence rose significantly above control when interstitial PO(2) fell to 2.9 +/- 0.5 mmHg (n = 13) and was not significantly different (2.4 +/- 0.5 mmHg) when the two variables were first averaged for all sites and then compared. Similar values were obtained using the abrupt change in rate of PO(2) decline as the criterion for critical PO(2). With a similar protocol, we determined that NADH rose significantly at a tissue site centered 30 micrometer from a collecting venule when intravascular PO(2) fell to 7.2 +/- 1.5 mmHg. The values for critical interstitial and critical intravascular PO(2) are well below those reported during free blood flow in this and in other muscle preparations, suggesting that oxygen delivery is regulated at levels well above the minimum required for oxidative metabolism. The extracellular critical PO(2) found in this study is slightly greater than previously found in vitro, possibly due to differing local conditions rather than a difference in metabolic set point for the mitochondria.The main purpose of this study was to determine the interstitial oxygen tension at which aerobic metabolism becomes limited (critical [Formula: see text]) in vivo in resting skeletal muscle. Using an intravital microscope system, we determined the interstitial oxygen tension at 20-μm-diameter tissue sites in rat spinotrapezius muscle from the phosphorescence lifetime decay of a metalloporphyrin probe during a 1-min stoppage of muscle blood flow. In paired experiments NADH fluorescence was measured at the same sites during flow stoppage. NADH fluorescence rose significantly above control when interstitial[Formula: see text] fell to 2.9 ± 0.5 mmHg ( n = 13) and was not significantly different (2.4 ± 0.5 mmHg) when the two variables were first averaged for all sites and then compared. Similar values were obtained using the abrupt change in rate of[Formula: see text] decline as the criterion for critical [Formula: see text]. With a similar protocol, we determined that NADH rose significantly at a tissue site centered 30 μm from a collecting venule when intravascular[Formula: see text] fell to 7.2 ± 1.5 mmHg. The values for critical interstitial and critical intravascular[Formula: see text] are well below those reported during free blood flow in this and in other muscle preparations, suggesting that oxygen delivery is regulated at levels well above the minimum required for oxidative metabolism. The extracellular critical[Formula: see text] found in this study is slightly greater than previously found in vitro, possibly due to differing local conditions rather than a difference in metabolic set point for the mitochondria.

Magnetic resonance imaging of tissue and vascular layers in the cat retina.

To report the visual resolution of multiple cell and vascular “layers” in the cat retina using MRI.

A Method for Chorioretinal Oxygen Tension Measurement

Purpose: To report an optical imaging system that was developed to measure oxygen tension (pO2) in the chorioretinal vasculatures. The feasibility of the system for the measurement of changes in pO2 separately in the retinal and choroidal vasculatures was established in rat eyes by varying the fraction of inspired oxygen and inhibiting nitric oxide activity. Methods: Our optical section phosphorescence imaging system was modified to provide quantitative measurements of pO2 separately in the retinal and choroidal vasculatures. A narrow laser line was projected at an angle on the retina after intravenous injection of an oxygen-sensitive probe (Pd-porphyrin), and phosphorescence emission was imaged. A frequency-domain approach allowed measurements of the phosphorescence lifetime by varying the phase relationship between the modulated excitation laser light and sensitivity of the imaging camera. Chorioretinal pO2 was measured while varying the fraction of inspired oxygen and during intravenous infusion of Nω-nitro-L-arginine (Nω-NLA), a nonselective nitric oxide synthase inhibitor. Results: The systemic arterial pO2 varied according to the fraction of inspired oxygen. The pO2 in the retinal and choroidal vasculatures increased as the fraction of inspired oxygen was increased. Compared with baseline, choroidal pO2 decreased during infusion of Nω-NLA, whereas the pO2 in the retinal vasculatures remained relatively unchanged. The choroidal pO2 decreased markedly with each incremental increase in Nω-NLA infusion rate, in the range 1–6 mg/min, and there was no additional change in the choroidal pO2 at Nω-NLA infusion rates above 6 mg/min. Conclusions: An optical method combining pO2 phosphorescence imaging with chorioretinal optical sectioning was established that can potentially be applied for better understanding of retinal and choroidal oxygen dynamics in physiologic and pathologic states.

Frequency Domain Imaging of Oxygen Tension in the Mouse Retina

Insufficient oxygen delivery and retinal hypoxia have been implicated as causal for the development of many devastating diseases of the eye, including diabetic retinopathy, glaucoma, and retinopathy of prematurity. The imaging of oxygen tension (PO2) using the phosphorescence lifetime measuring technique has received considerable attention in the literature and its potential for advancing our understanding of the oxygen dynamics in the retina is well recognized. It is also recognized that, as the number of transgenic and knockout mouse models displaying the characteristics of human retinal diseases rapidly increases, an ability to image PO2 in these very small eyes will likely be of great benefit. In this study, we describe and test new instrumentation for generating maps of PO2 in the mouse retina. These maps are generated from the phosphorescence lifetimes of injected palladium-porphyrin probes using a frequency-domain measurement.

EXPRESSION OF MYOGLOBIN IN THE TRANSGENIC MOUSE BRAIN

The main purpose of this study was to express human myoglobin in mouse brain neurons and investigate the effects of this expression on metabolism and blood flow using phosphorous (31P) NMR spectroscopy and NMR perfusion imaging. Transgenic mice expressing brain myoglobin were created using a cDNA sequence for human myoglobin placed under the transcriptional control of either a human platelet-drived grown factor polypeptide B (PDGF-B) promoter sequence or a rat neuron-specific enolase (NSE) promoter sequence. The presence of myoglobin having a functional, reduced-state, heme group was demonstrated by protein analysis and immunocytochemistry. Expression levels were highest in the hippocampus, cerebellum, and cerebral cortex. No gross morphological adaptations of neural tissue resulting from the expression were observed and no statistically significant differences in the energetic state, as measured by 31P NMR, or baseline cortical perfusion, as measured by an NMR perfusion imaging technique, were found.

Developing Strategies for Three-Dimensional Imaging of Oxygen Tension in the Rodent Retina

Insufficient oxygen delivery and retinal hypoxia have been implicated as causal in the development of many devastating diseases of the eye. While the two-dimensional imaging of retinal oxygen tension (PO2) has now been applied in a variety of different animal models, it is fundamentally a luminescence-based system lacking depth discrimination. However, mammalian retinal tissue is nourished by two distinct vascular beds, the retinal and the choroidal vasculatures, and they are exceedingly difficult to separate using traditional two-dimensional imaging strategies. Numerous studies have demonstrated that retinal and choroidal PO2 differ substantially. Therefore, the single PO2 value currently returned through data analysis cannot accurately represent the separate contributions of the choroidal and retinal vasculatures to the state of retinal oxygenation. Such a separation would significantly advance our understanding of oxygen delivery dynamics in these two very distinct vasculatures. In this study, we investigate new strategies for generating separate retinal and choroidal PO2 maps in the rodent retina using our existing phosphorescence-based lifetime imaging system.

Oxygen Delivery to the Retina and Related Visual Pathology

The survival of the retina and optic nerve is dependent on an adequate supply of oxygen and nutrients to fuel the metabolic processes that are vital for tissue homeostasis and primary visual function. Ocular diseases that affect the retina, including diabetic retinopathy, retinopathy of prematurity, glaucoma, and numerous proliferative retinopathies, are devastating diseases leading to major vision loss in humans. While the etiologies of these diseases are different, abnormalities in the delivery and consumption of oxygen are thought to be significant in all of them. Despite this common factor, quantitative studies that adequately correlate disease progression with changes in retinal oxygenation are lacking. In part, this is due to the almost complete absence of instrumentation for monitoring oxygenation in humans and the relatively few animal models that exhibit the full pathophysiology of these different retinal diseases.

Distinguishing between asthma and pneumonia through automated lung sound analysis

To help bridge the gap between common respiratory diagnosis and modern computer technology, we propose a system to record lung sounds in patients and digitally analyze them. This system distinguishes between asthma and pneumonia, two commonly misdiagnosed diseases.

Feasibility of two-dimensional color imaging in coronary vessels using a single-mode fiber catheter

This project seeks to combine the advantages of optical coherence tomography (OCT) and two-dimensional color imaging. The addition of color to OCT will allow cardiologists to have a more real-life image of the interior surface of the artery along with the high-resolution radial penetration of OCT. As of yet, no coronary color imaging has been done through a single fiber with, or independent of, an OCT system. It is hoped that using a single-mode fiber will help to alleviate the mechanical and safety limitations of angioscopy as well as adding to the information received from OCT. This new imaging mode, consisting of visible light imaging through a single-mode optical fiber, may allow not only for better detection, but also permit in vivo observation of lipid plaque histology and its role in causing myocardial infarctions.