[Show abstract][Hide abstract] ABSTRACT: 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.
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 Nomega-nitro-L-arginine (Nomega-NLA), a nonselective nitric oxide synthase inhibitor.
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 Nomega-NLA, whereas the pO2 in the retinal vasculatures remained relatively unchanged. The choroidal pO2 decreased markedly with each incremental increase in Nomega-NLA infusion rate, in the range 1-6 mg/min, and there was no additional change in the choroidal pO2 at Nomega-NLA infusion rates above 6 mg/min.
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.
Current Eye Research 05/2006; 31(4):357-66. DOI:10.1080/02713680600599446 · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To report the visual resolution of multiple cell and vascular “layers” in the cat retina using MRI.
T2- and diffusion-weighted MRI at 4.7 Tesla was performed. Layer-specific thickness, T2, spin density, apparent diffusion coefficient perpendicular (ADC⟂) and parallel (ADC∥) to the retinal surface were tabulated. T1-weighted MRI was acquired before and after intravenous administration of Gd-DTPA and subtraction images were obtained. Histology was performed for validation.
Three distinct “layers” were observed. The inner strip nearest to the vitreous (exhibiting large T2, ADC, spin density with Gd-DTPA enhancement) overlapped the ganglion cell layer, bipolar cell layer, and the embedded retinal vascular layer. The middle strip (exhibiting small T2, ADC, spin density without Gd-DTPA enhancement) overlapped the photoreceptor cell layer and the inner and outer segments. The outer strip (exhibiting large T2, ADC, spin density with Gd-DTPA enhancement) overlapped the tapetum and choroidal vascular layer. T2, spin density, ADC⟂ and ADC∥ of different “layers” were tabulated. The inner strip was slightly thicker than the other two strips. The total thickness, including neural and nonneural retina, was 358 ± 13μm (N = 6) by MRI and 319 ± 77 μm (N = 5) by histology.
MRI provides a noninvasive tool to study the retina with laminar specificity without depth limitation. J. Magn. Reson. Imaging 2006. Published 2006 Wiley-Liss, Inc.
Journal of Magnetic Resonance Imaging 04/2006; 23(4):465 - 472. DOI:10.1002/jmri.20549 · 3.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Advances in Experimental Medicine and Biology 02/2005; 566:173-8. DOI:10.1007/0-387-26206-7_24 · 1.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Advances in Experimental Medicine and Biology 02/2003; 530:331-45. DOI:10.1007/978-1-4615-0075-9_31 · 1.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 American journal of physiology 12/1999; 277(5 Pt 2):H1831-40. · 3.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A newly developed microscope using acousto-optic tunable filters (AOTFs) was used to generate in vivo hemoglobin saturation (SO2) and oxygen tension (PO2) maps in the cerebral cortex of mice. SO2 maps were generated from the spectral analysis of reflected absorbance images collected at different wavelengths, and PO2 maps were generated from the phosphorescence lifetimes of an injected palladium-porphyrin compound using a frequency-domain measurement. As the inspiratory O2 was stepped from hypoxia (10% O2), through normoxia (21% O2), to hyperoxia (60% O2), measured SO2 and PO2 levels rose accordingly and predictably throughout. A plot of SO2 versus PO2 in different arterial and venous regions of the pial vessels conformed to the sigmoidal shape of the oxygen-hemoglobin dissociation curve, providing further validation of the two mapping procedures. The study demonstrates the versatility of the AOTF microscope for in vivo physiologic investigation, allowing for the generation of nearly simultaneous SO2 and PO2 maps in the cerebral cortex, and the frequency-domain detection of phosphorescence lifetimes. This class of study opens up exciting new possibilities for investigating the dynamics of hemoglobin and O2 binding during functional activation of neuronal tissues.
[Show abstract][Hide abstract] ABSTRACT: All of the variables regulating the delivery of oxygen to specific tissues have not been identified. However, O2 capacity, blood flow, and vessel diameter, are coordinated in concert to optimize tissue oxygenation. Experimental models studied to date indicate that the set points for muscle blood flow are well above those needed to maintain oxidative metabolism. Thus, additional mechanisms must exist. A possible point of control is the supply of O2 to arterioles which can regulate capillary blood flow. This hypothesis has important implications for the development of red cell substitutes.
[Show abstract][Hide abstract] ABSTRACT: Recessed cathode O2 microelectrodes were used to measure spatially detailed oxygen tension (PO2) gradients in the vitreous humor near the cat retina. Measurement sites (n = 41 in 8 cats) included single arterioles and venules and parallel vessel pairs. Mean vitreous PO2 was 37.9 +/- 1.5 (SE) Torr. Close to the retinal surface (approximately 200 microns), PO2 was found to be both higher and lower than the vitreous PO2, depending on the proximity of the microelectrode tip to retinal vessels. Both positive (inward) and negative (outward) O2 fluxes (JO2) were measured, consistent with the anatomy and expected boundary conditions in the eye. The PO2 at the closest approach above arterioles was 55.2 +/- 2.3 Torr, significantly higher than in the vitreous (P < 0.0001). All arterioles had outward JO2 with an overall mean of -2.58.10(-6) ml O2/sec/cm2. Some of the venules were also losing O2, but at much lower rates than arterioles. Several venules were gaining O2. Countercurrent transport (A-V shunting) was also seen between vessel pairs. Our experimental results allow theoretical predictions to be made for the axial drop in blood PO2 along an arteriole as a function of blood flow.
Microvascular Research 03/1993; 45(2):134-48. DOI:10.1006/mvre.1993.1013 · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A newly developed phosphorescence imaging technique was used to generate two-dimensional maps of intravascular oxygen tension (PO2) in the optic nerve head (ONH) and retina of the cat to study the effects of acute moderate increases in intraocular pressure (IOP) on the ONH and retinal PO2. Both the ONH and retinal PO2 were remarkably well maintained as the IOP increased; hypoxia developed only after the blood flow to the eye was stopped. Because ONH hypoxia was not observed during IOP elevation, a lack of oxygen may not be a major cause of glaucomatous damage, although the effects of chronically elevated IOP on the PO2 remain to be evaluated. Because this imaging technique was noninvasive and required only a small bolus injection of a nontoxic oxygen probe, the authors anticipate that it will find significant application in the study of many ocular vascular diseases and glaucoma.
[Show abstract][Hide abstract] ABSTRACT: Laser Doppler flowmetry (LDF) is a technique that measures relative average velocity, number and flux (number times velocity) of red blood cells in a tissue. In this paper, we demonstrate its application in the optic nerve head tissue, describe the laser delivery and light scattering detection schemes and investigate the effect of the distance between the sites of illumination and detection. We also provide evidence that the flow measured by LDF varies linearly with actual blood flow in the optic nerve and examine the question of the depth of the sampled volume. Experiments in anesthetized cats illustrate potential applications which make use of the high temporal resolution of LDF. These include the response of blood flow to changes in the composition of the breathing gases and changes induced by neuronal stimulation with multiple and single flashes.
Experimental Eye Research 10/1992; 55(3):499-506. DOI:10.1016/0014-4835(92)90123-A · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The oxygen tension in the vessels of the retina and optic nerve head has been measured noninvasively with a new phosphorescence imaging method. A phosphorescent oxygen-dependent probe, injected into the bloodstream of cats, was excited with a flash of light and the phosphorescence lifetime of the probe was measured. A simple Stern-Volmer relationship was used to convert lifetime to oxygen tension, and two-dimensional maps of intravascular oxygen tension were produced. We describe the equipment and the methodology for obtaining oxygen maps.
[Show abstract][Hide abstract] ABSTRACT: A new ocular micromanipulator for introducing a microelectrode into the mammalian eye is described. Included is a technical description of the device, incorporating arched tracks and DC motors to provide for angular motion around a scleral entrance point. Applications for in vivo measurements of local PO2 in the retina and vitreous of miniature pigs and cats are shown. The new design features of this micromanipulator allow for smooth microelectrode movements over large retinal areas, facilitating the rapid collection of data in many different areas of the retina and vitreous.
Experimental Eye Research 01/1992; 53(6-53):723-727. DOI:10.1016/0014-4835(91)90107-P · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effect of diffuse luminance flicker stimulation of a large area (approximately 30 degrees diameter) on red blood cell flux (F) in the optic nerve head was measured in the anesthetized cat. F increased markedly during sustained flicker. The F-response to the initiation and cessation of the stimulation was found to occur within a few seconds. Upon sustained stimulation, the increase in F reached a plateau within approximately 2 min. Its level depended upon the intensity, frequency and wavelength of the stimulation and the state of adaptation of the retina. This stimulus offers a new and powerful means of investigating blood flow regulation in the optic nerve head (ONH).
[Show abstract][Hide abstract] ABSTRACT: Laser Doppler velocimetry was performed on retinal vessels of a cat using a linearly polarized He-Ne laser as the incident beam. The diameter of the vessels measured was <120 microm. Measurements show that if double transmission of the laser light through a given retinal vessel can be prevented, the Doppler shift power spectra have the shape theoretically expected from calculations based on a single scattering model and parabolic velocity profile of the red blood cells.