Fuminori Goda

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.

Effect on regrowth delay in a murine tumor of scheduling split-dose irradiation based on direct pO2 measurements by electron paramagnetic resonance oximetry

Tumor reoxygenation after irradiation may contribute to a tumors response to subsequent doses of radiation. The timing of reoxygenation in RIF-1 murine tumors was determined using electron paramagnetic resonance (EPR) oximetry with intratumoral implantation of an oxygen-sensitive paramagnetic material (India ink) to monitor the pO2 in individual murine tumors before, during and after three different irradiation schemes. Radiation was given as a single 20-Gy dose or was split into two 10-Gy doses where the second dose of radiation was delivered at the minimum postirradiation tumor pO2 (24-h interval, hypoxic group) or where the second dose of radiation was delivered after reoxygenation had occurred (72-h interval, oxygenated group). The end point for tumor response was time taken to reach double the volume at the time of treatment. There were significantly longer tumor doubling times in the oxygenated compared to the hypoxic group, indicating that the measured changes in pO2 reflected changes in tumor radiosensitivity. A 24-h interval between doses resulted in a delay of reoxygenation in the tumors, while a 72-h interval resulted in a second cycle of hypoxia/reoxygenation. Our results suggest that repeated direct measurements of pO2 in tumors by EPR oximetry could be useful in timing radiation doses to achieve improved local control of tumors.

The pO2 in a Murine Tumor after Irradiation: An In Vivo Electron Paramagnetic Resonance Oximetry Study

Using electron paramagnetic resonance (EPR) oximetry with the oxygen-sensitive paramagnetic material, fusinite, we have measured the partial pressure of oxygen (pO2) in the mouse mammary adenocarcinoma MTG-B. The average pO2 in untreated tumors was low (about 5 mm Hg) and decreased with tumor growth. Magnetic resonance imaging and histological examination were used to localize the position of the fusinite with respect to tumor margins and vascularization. The pO2 was generally higher in the periphery than in the center of the tumors, but there was considerable variation among tumors both during normal growth and after radiation treatment. After a single 20-Gy dose, a characteristic pattern of change in tumor pO2 was observed. In irradiated tumors, there was an initial reduction in pO2 (minimum occurred 6 h postirradiation) which was followed by a transient increase in pO2 to levels higher than the preirradiation pO2 (maximum occurred 48 h postirradiation). This work demonstrates postirradiation changes in pO2 of potential radiobiological significance. Compared to other oxygen assessment techniques, EPR oximetry is very useful because it can assess pO2 in the same region of the tumor over the course of tumor growth and during response to treatment. Thus EPR could be used to identify potentially radioresistant tumors as well as to identify tumors with slow reoxygenation.

Effect of Anesthesia on Cerebral Tissue Oxygen and Cardiopulmonary Parameters in Rats

General anesthesia is known to alter cardiopulmonary and hematological parameters which affect tissue oxygenation. However, the effect of various types of anesthetics on the relationship between brain tissue pO2 and these physiologic parameters is still largely unknown.

Comparisons of Measurements of pO2 in Tissue In Vivo by EPR Oximetry and Micro-Electrodes

Polarographic micro-electrode measurements are very useful for measuring pO2 in vivo, especially for measurements of the variation of pO2 within a tumor (1,2,8). This method has several advantages, including: it is the only direct method currently in extended use in the clinical setting; it can provide data on microscopic heterogeneity; and it is fairly widely available. While the micro-electrode method has become a type of “gold standard” for measurement of pO2 in tissues, it has some limitations and disadvantages: it can be technically difficult; it has limited resolution at the very low levels of pO2 that are important for many clinically relevant processes; and it can perturb the tissues significantly, especially when used in repeated studies to monitor pO2 in tissues over time. Repeated measurements are especially desirable to follow the effect on tissue pO2 after treatment with some drugs (e.g. anti-cancer drugs and anesthetics) and radiation, the effects of acute and chronic ischemia, and changes in respiratory factors. Electron paramagnetic resonance (EPR) oximetry appears to offer some complimentary advantages for such studies: it can monitor pO2 continuously and/or repeatedly at the exactly the same localized area in tissue in vivo without the need for anesthesia; it can resolve small differences in pO2 even at the very low levels that occur pathophysiologically; and it can be used in a variety of settings.

Measurements of pO2 in Vivo, Including Human Subjects, by Electron Paramagnetic Resonance

The purpose of this paper is to provide an illustrative description of the current state of development of the use of electron paramagnetic resonance (EPR, or completely equivalently, electron spin resonance or ESR) to measure the partial pressure of oxygen (pO2) in tissues in vivo under physiological conditions. This summary is based on published and unpublished results from our laboratory (1–7) and does not attempt to describe the results of other laboratories which also are working along related lines (8–10). The pertinent features of our technique are illustrated. We also consider the current limitations of the technique and likely developments in the near future. Our evaluation is that: this technique now is suitable for immediate use in small animals; within a short period of time instruments will be available facilitating its use in larger animals; and preliminary studies are imminent in human subjects (7).

Potential for EPR Oximetry to Guide Treatment Planning for Tumors

A major contributing factor in the failure of solid tumors to be locally controlled by radiation therapy (RT) is the relative radiation resistance of hypoxic cells of tumors compared to well-oxygenated cells. Recently clinical studies have established the presence of hypoxic regions in human tumors (1,2). Further studies confirmed that relatively high tissue pO2 in human tumors correlated with positive outcomes of radiation therapy and that poor outcomes were associated with tumors with low pO2 (3–6). As a result of available studies, the conclusion has been drawn that the effective level of oxygenation in individuals could not be predicted based on tumor type, histology, staging, or even size, but had to be measured (7). With the development of methods to measure the pO2 in tumors (8), it now seems feasible to determine if this parameter, which can be of crucial importance in radiation therapy, can be used to improve treatment by permitting individualized optimization of therapy on the basis of the pO2 in the tumor.

Intrarenal pO2 Measured by EPR Oximetry and the Effects of Bacterial Endotoxin

This study used Electron Paramagnetic Resonance (EPR) oximetry to detect the signal arising from oxygen-sensitive crystals of Lithium phthalocyanine (LiPC) implanted in the cortex and outer medulla of an isolated perfused rat kidney. Kidneys with implanted crystals were placed beneath the surface detector of an L-band spectrometer, and an additional gradient was induced between the poles of the magnet so as to separate the signals arising from each region, allowing simultaneous measurement of the partial pressure of oxygen (pO2) at two locations within the same organ. In control kidneys, the pO2 in the cortex was 96.9±7 and that of the outer medulla 11.0±4 mmHg. We found that perfusion pressure could be increased with little effect on the pO2 of the outer medulla. At a critical point (≈140 mmHg) however, pO2 in this region was markedly increased and was accompanied by a decrease in vascular resistance. When kidneys treated in this way were then given L-NMMA (an inhibitor of nitric oxide synthase), the pO2 in the outer medulla returned to baseline values, presumably by blocking nitric oxide-induced vasodilation. Inclusion of Lipopolysaccharide (LPS) into the perfusion media of control kidneys resulted in a decrease in the pO2 in the cortical region and an increase in the outer medullary region. When L-NMMA was given prior to administration of LPS, the changes in pO2 were prevented. Based on these results, we have developed a model which can account for these observations. It indicates that re-distribution of blood (and hence oxygen) within the kidney may have an important role in altering the solute (and oxygen) gradient early during the septic episode.

In vivo EPR spectroscopy

This chapter is intended to provide a brief overview of the principles of electron paramagnetic resonance (EPR, or completely equivalently electron spin resonance, ESR) spectroscopy applied to living animals. It attempts to indicate especially those areas in which this approach is likely to be of value because it can provide useful information that cannot be provided as well by other approaches. As a matter of convenience the descriptions are drawn principally from the authors’ laboratory but it should be noted that there are a number of laboratories around the world, especially in Japan, which are also actively pursuing these developments. Because of the need for brevity in this volume, the coverage is illustrative rather than comprehensive but this fits well with the aim of the book which is to provide a review that will be useful for the longer term rather than only a review of the current state of development.

Pharmacokinetics of the nitroxide PCA measured by in vivo EPR

PCA (2,2,5,5-tetramethylpiperidine-1-oxyl-3-carboxylic acid) is a relatively stable free radical which has been shown to be useful as a contrast agent for nuclear magnetic resonance imaging and as an imaging/spectroscopy agent for EPR. In an effort to determine the role of the liver and kidney in the pharmacokinetics of PCA, using low frequency in vivo EPR spectroscopy, we followed the clearance of PCA after intravenous injection in mice: under normal conditions, with a restricted blood supply to the kidneys, after exposure to an acute hepatotoxin CCl4, and after exposure to lipopolysaccharide (endotoxin). The observed pharmacokinetics fit a two-component model. The fast component was dramatically affected when the renal vessels were restricted, while CCl4 and endotoxin had a smaller but significant effect. The half times of the slow components were not significantly different (p>0.05) in the groups treated by renal blood flow occlusion, CCl4, or LPS, compared with the control group. In conclusion, we find that the pharmacokinetics of PCA need to be completely described in term of a two component model: the fast component of the decay is mainly due to the elimination by the kidneys and also is affected by the time for the initial distribution; the slow component is related to the bioreduction of the nitroxide. In addition to the liver other tissues can also effectively metabolize PCA. The effect of oxygen on the rate of metabolism is modest at most.