Development and evaluation of biocompatible films of polytetrafluoroethylene polymers holding lithium phthalocyanine crystals for their use in EPR oximetry.

Electron paramagnetic resonance (EPR) oximetry is a powerful technology that allows the monitoring of oxygenation in tissues. The measurement of tissue oxygenation can be achieved using lithium phthalocyanine (LiPc) crystals as oxygen reporters. In order to have biocompatibility for the sensing system and to assure long-term stability in the responsiveness of the system, we developed films of Teflon AF 2400 with embedded LiPc crystals. These systems can be used as retrievable inserts or parts of an implantable resonator or catheter. Atomic force microscopy studies revealed that the surface of the films was regular and planar. The response to oxygen of the sensor (EPR linewidth as a function of pO(2)) remained unchanged after implantation in mice, and was not affected by sterilization or irradiation. The use of resonators, holding LiPc embedded in Teflon AF 2400, implanted in the gastrocnemius muscle of rabbits allowed the monitoring of oxygen during several weeks. Several assays also demonstrated the biocompatibility of the system: (1) no hemolytic effect was noted; (2) no toxicity was found using the systemic injection test of extracts; (3) histological analysis in rabbit muscle in which the films were implanted for 1 week or 3 months was similar to standard polyethylene biocompatible devices. These advanced oxygen sensors are promising tools for future pre-clinical and clinical developments of EPR oximetry. These developments can be applied for other applications of biosensors where there is a need for oxygen permeable membranes.

High spatial resolution multi-site EPR oximetry. The use of convolution-based fitting method.

We describe a new method to enhance the spatial resolution of multi-site electron paramagnetic resonance (EPR) oximetry. The method is suitable for any shape (density distribution function) of a solid paramagnetic material implanted in tissue. It corrects distortions of lineshapes caused by the gradient and thus overcomes limitations of previous multi-site EPR oximetry methods that restricted the ratio of the particle size to the distance between sites. The new method is based on consecutive applications of magnetic field gradients with the same direction but with a different magnitude and uses a convolution-based fitting algorithm to derive Lorentzian EPR linewidths of each individual peak of the EPR spectrum. The method is applicable for any particulate EPR oxygen sensitive materials whose EPR spectra can be approximated by a Lorentzian function or a superposition of Lorentzian functions. By incorporating this model of the lineshape in the data processing, we are able to decrease significantly the number of parameters needed for the calculations and to recover the oxygen concentration, even from quite noisy spectra. We (i) describe our method and the data-processing algorithm, (ii) demonstrate our approach in model and in vivo experiments, and (iii) discuss the limitations.

Response to radioimmunotherapy correlates with tumor pO2 measured by EPR oximetry in human tumor xenografts.

The efficacy of radiation treatment depends upon local oxygen concentration. We postulated that the variability in responsiveness of tumor xenografts to a fixed dose of radioimmunotherapy might be related to the tumor pO2 at the time that radioimmunotherapy was administered. We evaluated the growth of xenografts of CALU-3 tumors, a non-small cell lung carcinoma, in response to an 8.9-MBq dose of 131I-RS-7-anti-EGP-1 and correlated tumor growth rate with initial tumor pO2 measured by EPR oximetry. The greatest growth delay in response to radioimmunotherapy had the highest initial pO2, and the fastest-growing tumors had the lowest initial pO2. We then determined the dynamic effect of radioimmunotherapy on tumor pO2 by serial measurements of pO2 for 35 days after radioimmunotherapy. This information could be important for ascertaining the likelihood that a tumor will respond to additional doses as part of a multiple dose scheme. Serial tumor pO2 measurements may help identify a window of opportunity when the surviving tumor regions will be responsive to a second round of radioimmunotherapy or a second therapeutic modality such as chemotherapy or an anti-vascular agent. After radioimmunotherapy, there was an increase in tumor pO2 followed by a decrease below initial levels in most mice. Thus defined times may exist when a tumor is more or less radiosensitive after radioimmunotherapy.

Superoxide production by phagocytosing macrophages in relation to the intracellular distribution of oxygen.

We simultaneously measured the concentration of oxygen ([O2]) within the phagosomal and extracellular compartments of macrophages. By combining electron paramagnetic resonance (EPR) oximetry techniques with that of spin-trapping, we found that a significant difference in oxygen concentration ([O2]) exists between these two compartments and we were able to monitor (1) how [O2] in the extracellular compartment and the rate of mitochondrial consumption affected this difference in [O2], and (2) to what extent this gradient of [O2] influenced production of reactive oxygen species by phagosomes. Under conditions where the [O2] in the inflowing gas was high (210 microM; air), the [O2] in the extracellular and phagosomal compartments was 180 and 141 microM, respectively. This was sufficient to maintain maximum superoxide production in these cells. When extracellular [O2] was reduced to 84 or 36 microM, the [O2] in phagosomes within the cells (31.7 and 7.7 microM, respectively) was too low to maintain superoxide production by the NADPH-oxidase system within the phagosomes. The [O2] in the extracellular compartments of these samples, however, was always sufficient to maintain superoxide production by phagosomes at the cell surface. Our findings suggest that the distribution of oxygen surrounding and within macrophages can influence their ability to perform microbicidal and tumoricidal functions, even at an [O2] in the media that appears to be adequate.

Acute hemodynamic and coronary circulatory effects of experimental autoimmune myocarditis.

Myocarditis and progression to cardiomyopathy is associated with focal spasm and reperfusion of the coronary microcirculation. Experimental autoimmune myocarditis (EAM), induced with cardiomyosin peptide-specific T cells in Lewis rats, was hypothesized to cause acute hemodynamic and coronary vasculature changes. Fifteen experimental animals (5 each at 1, 2, and 3 weeks after T-cell injection) and eight controls were studied using the constant pressure variant of the isolated heart. Coronary resistant decreased while coronary flow increased (P < 0.05) in EAM hearts after the first week. Rate-pressure product, +dP/dt and -dP/dt, decreased while the heart/body weight ratio increased (P < 0.05) compared with controls at 1 week but not at 2 or 3 weeks. Mean local myocardial PO2, which reflects local oxygen delivery and consumption, and MVO2 were not different for EAM hearts. However, compared with controls EAM myocardial PO2 varied more widely and was often beyond the usual range, suggesting the occurrence of localized hypoxic and hyperoxic areas. In summary, after the first week there was a significant decrease in coronary resistance in the EAM animals, which required higher flow to maintain a similar perfusion pressure. These changes in coronary resistance and flow along with the heterogeneity and extremes of local myocardial PO2 levels without a significant change in MVO2 may be explained by postulating development of low-resistance, high-flow hyperoxic areas which steal flow, thus causing hypoxia in other areas.

Are there significant gradients of pO2 in cells?

It is widely recognized that the intracellular oxygen tension (pO2) plays an important role in cellular function and metabolism. In experimental and theoretical consideration involving the role of the pO2 the values that are used usually are those for the pO2 at the exterior of cells, because these values can be more readily measured. Such an approach is based on the assumption that the intracellular pO2 is very similar to the extracellular pO2 because oxygen freely diffuses across cell membranes and within cells, at a rate that is similar to that in the extracellular media. For the past several years we have been developing and applying electron paramagnetic resonance (EPR) techniques to test directly whether there are intracellular gradients of pO2 and if so, where they occur and what factors determine them. We previously reported significant gradients between the average pO2 in the intracellular and extracellular compartments in cell suspensions (Glockner 1989). More recently we developed techniques that enabled us to measure simultaneously the concentration of oxygen within a specific compartment, the phagosomes of activated macrophages, and the extracellular compartment and found gradients of up to 48 microM under some conditions (James 1995). The precise mechanism of the intracellular-extracellular oxygen gradient remains uncertain. The possibilities include that the diffusion of oxygen is not as free as assumed (e.g. that the cell membrane can act as a barrier) and the occurrence of active transport of O2 out of the cells. We report here on the use of a simple theoretical approach to evaluate the values of three key parameters which might account for the observed intracellular-extracellular oxygen measurements: (1) oxygen consumption; (2) the diffusion coefficient of oxygen in the cytosol; (3) the solubility of oxygen in the cytosol. We used two different models for the relationship between the oxygen consuming compartment (assumed to be primarily the mitochondria) and the intracellular compartment in which the measurements were made (especially phagosomes): uniform and non-uniform distribution of the mitochondria. Using these models and consensus values from the literature, we were unable to account for the experimentally observed differences in pO2 between the intracellular and extracellular compartments. Also we found that with the variation of any one parameter we could not plausibly account for the measurements made in the phagosomal and extracellular compartments. There are at least three logical possibilities to account for these results: 1) this methodology is erroneous and/or produces artifacts in the system resulting in invalid results; 2) the observation of a gradient in oxygen concentration between these two compartments arises from significant simultaneous variations of more than one of the critical parameters which are used conventionally to calculate potential gradients in pO2; 3) there is another factor not considered in the model which accounts for the observation (e.g. active transport; significantly higher than expected barriers to oxygen diffusion in the membrane).

Myocardial oxygen tension in isolated erythrocyte-perfused rat hearts and comparison with crystalloid media.

The isolated heart, typically perfused with crystalloid media equilibrated with >/=95% O2 to ensure adequate myocardial oxygen tension, is commonly used to study cardiac function. When hemoglobin is available for oxygen transport, equilibration with 21% O2 is considered adequate to meet metabolic demands. This study presents the measurement of myocardial pO2 in isolated hearts perfused with an erythrocyte suspension. Baseline myocardial pO2 in erythrocyte-perfused hearts was 16.4+/-3.5 mmHg (mean+/-s.e.). When compared to previous measurements of myocardial pO2 in isolated hearts perfused with crystalloid media, the use of erythrocyte suspensions resulted in a 10-fold lower level of myocardial pO2, while avoiding very low and high values. The standard use of 95% oxygen with crystalloid results in myocardial levels of oxygen far above those usually found in the presence of hemoglobin and room air.

Gloxy: an oxygen-sensitive coal for accurate measurement of low oxygen tensions in biological systems.

This paper describes the characteristics of a new oxygen sensitive, paramagnetic material that has some significant advantages for measurements of tissue pO2 by in vivo EPR. This paramagnetic component of Welsh coal, termed "gloxy" was found to have valuable EPR features that allow accurate measurement of low oxygen tensions in vivo; these include large oxygen-dependent changes in linewidth, a high number of paramagnetic spin centers (resulting in high signal amplitude), and stability in tissue allowing repeated pO2 measurements to be made in vivo with high precision. Renal pO2 was measured deep in the medulla region of isolated perfused kidneys and found to be lower than that in the cortex (1.7 +/- 0.05 and 7.1 +/- 0.3 mm Hg, respectively). The quality of the EPR signal obtained from the renal outer medulla and also from tumors in mice was such that the pO2 measurements were obtained with a precision of +/-3% of the measured pO2 (Kidney: 1.7 +/- 0.05 mmHg; Tumor: 1.37 +/- 0.04 mmHg). In vitro tests on the viability of cells and in vivo studies using Gloxy demonstrate the stability and inertness of this oxygen-sensitive material.

Myocardial oxygen tension and capillary density in the isolated perfused rat heart during pharmacological intervention.

Oxygen is essential for normal cardiac function and plays an important role in cardiac regulation. Electron paramagnetic resonance (EPR) oximetry appears to have some significant advantages for measuring oxygen tension (pO2) in the beating heart. This study presents the serial measurement of myocardial pO2 by EPR oximetry in the isolated crystalloid perfused heart during treatment with different cardioactive drugs: dobutamine, metoprolol, verapamil, vasopressin, and N omega-Nitro-L-Arginine Methyl Ester (L-NAME). Baseline myocardial pO2 was 176 +/- 14 mmHg (mean +/- S.E.). Myocardial capillary density in the intact contracting heart was calculated to be 2300 +/- 100 mm-2, using local myocardial pO2 and a cylindrical model for oxygen diffusion in tissue. Each drug had characteristic effects on myocardial pO2, myocardial oxygen consumption (MVO2), and capillary density. Metoprolol and verapamil increased myocardial pO2 by 51% and 18%, respectively, dobutamine decreased myocardial pO2 by 84% while vasopressin and L-NAME had no significant effect on myocardial pO2. Metoprolol and verpamil decreased MVO2 by 9% and 56%, respectively, while dobutamine increased MVO2 by 59%. A quantitative comparison of effects on the capillary bed based on changes in myocardial pO2 and MVO2 was made. Metoprolol and verapamil had opposite effects on the capillary bed. Verapamil decreased myocardial capillary density by 39%, while capillary density increased by 10% (n.s.) with metoprolol. Data following perfusion without drug is also presented. We conclude that: 1) The application of EPR oximetry with LiPc provides dynamic evaluation of local myocardial pO2 in the contracting heart. 2) Using a cylindrical model of oxygen delivery and diffusion in tissue, these data may be used to describe the changes of capillary density during pharmacological interventions.

Effect of repetitive ischemia on myocardial oxygen tension in isolated perfused and hypoperfused rat hearts.

The objective of this study was to determine the effects of repetitive ischemia on myocardial oxygen tension (pO2), consumption, and delivery in crystalloid normoperfused (perfusion pressure>70 mmHg) and hypoperfused (perfusion pressure approximately 50 mmHg) constant flow isolated rat hearts. EPR oximetry with lithium phthalocyanine was used to measure myocardial pO2. Baseline myocardial pO2 (means +/- SE) was 185 +/- 13 mmHg (normoperfused) and 162 +/- 14 mmHg (hypoperfused). Myocardial pO2 fell to < 1 mmHg during no-flow ischemia. After recovery from repetitive ischemia, myocardial pO2 and coronary resistance increased significantly in all hearts; oxygen consumption and left ventricle work decreased in normoperfused hearts, although not significantly compared with controls, and did not change significantly in hypoperfused hearts. Increased myocardial pO2 in the normoperfused group may be due to decreased oxygen consumption and/or increased local delivery, while increased myocardial pO2 in the hypoperfused hearts is due to increased local oxygen delivery.

Endotoxin-induced changes in intrarenal pO2, measured by in vivo electron paramagnetic resonance oximetry and magnetic resonance imaging.

Electron Paramagnetic Resonance (EPR) oximetry was used to measure tissue oxygen tension (pO2-partial pressure of oxygen) simultaneously in the kidney cortex and outer medulla in vivo in mice. pO2 in the cortex region was higher compared to that in the outer medulla. An intravenous injection of endotoxin resulted in a sharp drop in pO2 in the cortex and an increase in the medulla region, resulting in a transient period of equal pO2 in both regions. In control kidneys, functional Magnetic Resonance (MR) images showed the cortex region to have high signal intensity (T2*-weighted images), indicating that this region was well supplied with oxygenated hemoglobin, whereas the outer medulla showed low signal intensity. After administration of endotoxin, we observed an immediate increase in signal intensity in the outer medulla region, reflecting an increased level of oxygenated blood in this region. Pretreatment of mice with NG-monomethyl-L-arginine prevented both the changes in tissue pO2 and distribution of oxygenated hemoglobin, suggesting that localized production of nitric oxide has a critical role to play in renal medullary hemodynamics. In combining in vivo EPR with MR images of kidneys, we demonstrate the usefulness of these techniques for monitoring renal pO2 and changes in the distribution of oxygen.

Myocardial oxygen tension and relative capillary density in isolated perfused rat hearts.

Oxygen plays an important role in cardiac function. Many methods have been applied to measure tissue oxygen tension (PO2). Electron paramagnetic resonance (EPR) oximetry appears to have some significant advantages for use in the beating heart. This study presents the serial measurement of myocardial PO2 by EPR oximetry in the isolated crystalloid perfused heart during changes of influent PO2, coronary flow rate, oxygen consumption and end-diastolic pressure. Baseline myocardial PO2 was 198 +/- 12 mmHg (mean +/- S.E.). Myocardial PO2 increased as expected with increased delivery (concentration or flow) or decreased consumption. With increasing flow rate, myocardial PO2 increased in a sigmoid fashion. A critical flow or pressure was reached when myocardial PO2 rapidly increased to a higher level. Increased left ventricular end-diastolic pressure caused local vascular compression and resulted in a decrease of myocardial PO2. Myocardial capillary density in the intact contracting heart was calculated to be 2300 +/- 110/mm2, using local myocardial PO2 and a cylindrical model for oxygen diffusion in tissue. Relative capillary density did not change with mild to moderate hypoxia, increased with increasing flow and increasing oxygen consumption and decreased with elevated diastolic pressure. We conclude that the application of EPR oximetry with LiPc to the isolated heart provides accurate and dynamic evaluation of local myocardial PO2 in the contracting heart. Using various models of oxygen delivery and diffusion in tissue, these data may also be used to serially follow capillary density.

Intraphagosomal oxygen in stimulated macrophages.

A new electron paramagnetic resonance (EPR)-based method was developed to obtain selective information on pO2 in a specific intracellular compartment (phagosomes). This method did not require the use of a broadening agent thereby eliminating one of the potential sources of experimental error with EPR oximetry. An oxygen-sensitive probe (4-(Trimethylammonium) 2,2,6,6-tetramethylpiperidine-d17-1-oxyl iodide (d-Cat1)) which has a net positive charge, was incorporated selectively into the phagosomes of macrophages stimulated with zymosan. Extracellular oxygen was measured by addition of a neutral nitroxide (4-oxo-2,2,6,6-tetramethylpiperidine-d16-1-oxyl (15N PDT)) to this same sample. Measurements based on EPR linewidths showed the average intraphagosomal oxygen concentration to be 11.2 +/- 3.4 microM lower than that measured from the extracellular compartment when the sample was perfused with air, and this was increased on stimulation of mitochondrial consumption or by increasing the oxygen concentration in the extracellular compartment. These experiments provide what we believe to be the first reported measurements of the oxygen concentration in a specific intracellular location (intraphagosomal) and its comparison with the oxygen concentration in the extracellular space. The observed gradient cannot be explained in terms of known coefficients of diffusion, and these results are consistent with previous reports that a gradient in oxygen concentration can occur between the average intracellular and extracellular concentration of oxygen.

The effects of endotoxin on oxygen consumption of various cell types in vitro: an EPR oximetry study.

We have studied the effects of bacterial endotoxin on the oxygen consumption of a variety of target cells, and found that the rate of utilization of oxygen by treated cells was decreased in a time- and dose-dependent manner. Precise EPR measurement of oxygen concentrations enabled us to demonstrate that this effect was linked to mitochondrial dysfunction and was particular to each cell type. Such detailed knowledge on oxygen utilization by viable whole cells and the varied effects of endotoxin are as yet undocumented. Oxygen consumption was shown to decrease quite markedly in CHO cells and kidney cells from the cortex region. Cells from the kidney medulla region had lower baseline consumption and were stimulated to increased levels of oxygen consumption on addition of similar doses of endotoxin. Macrophages exhibited a dual response in that in addition to inhibiting mitochondrial oxygen consumption, endotoxin pretreatment primed these cells to exhibit an enhanced oxidative burst on stimulation with Zymosan. These results show that endotoxin has a direct effect on normal cellular oxygen consumption and is an important parameter that must be considered when following the early effects on cells and tissues during the septic syndrome.