Oxygen effects on the EPR signals from wood charcoals: experimental results and the development of a model.

Charcoals prepared from certain tropical woods contain stable paramagnetic centers, and these have been characterized by EPR spectroscopy in the absence and presence of oxygen. The EPR-detectable spin density has been determined, as has been the temperature- and frequency-dependence of the oxygen broadening of the EPR signal, which is orders of magnitude larger than that observed with other materials, such as lithium phthalocyanine. Three Lorentzian components are required to fit the char EPR spectrum in the presence of oxygen, and the oxygen-dependence of the line width, intensity, and resonance position of the three components have been quantified. These results and the properties of porous carbonaceous materials are used to develop a model to explain the effect of oxygen on the char EPR spectral properties. The model is based on oxygen adsorption on the char surface according to a Langmuir isotherm and a dipolar interaction between the paramagnetic adsorbed gas and the charcoal spins. The three EPR components are correlated with the three known classes (sizes) of pores in charcoal, with the largest line broadening attributed to dipolar relaxation of spins in micropores, which have a larger specific surface area and a higher concentration of adsorbed oxygen. An attenuated, but similar, EPR response to oxygen by chars when they are immersed in aqueous solution is attributed to water competition with oxygen for adsorption on the char surface.

The effect of oxygen therapy on brain damage and cerebral pO(2) in transient focal cerebral ischemia in the rat.

We examined the effect of hyperbaric oxygen (HBO) and normobaric oxygen (NBO) on neurologic damage and brain oxygenation before and after focal cerebral ischemia in rats. A middle cerebral artery occlusion (MCAO)/reperfusion rat model was used. The rats were sacrificed 22 h after reperfusion, and the infarct volume was evaluated. In study A, HBO (2.0 ATA), NBO (100% oxygen) and normobaric air (NBA) were each administered for 60 min in five different rat groups. The sizes of the infarcts after HBO and NBO applied during ischemia were 8.8 +/- 2.8% and 22.8 +/- 3.7% respectively of the ipsilateral non-occluded hemisphere. The infarct size after HBO applied during ischemia was statistically smaller than for NBO and NBA exposure (p < 0.01). In study B, cerebral pO(2) was measured before and after MCAO and HBO exposure (2.0 ATA for 60 min) in six rats using electron paramagnetic resonance (EPR) oximetry. The pO(2) in the ischemic hemisphere fell markedly following ischemia, while the pO(2) in the contralateral hemisphere remained within the normal range. Measurements of the pO(2) performed minutes after HBO exposure did not show an increase in the ischemic or normal hemispheres. The mean relative infarct size was consistent with the changes observed in study A. These data confirm the neuroprotective effects of HBO in cerebral ischemia and indicate that in vivo EPR oximetry can be an effective method to monitor the cerebral oxygenation after oxygen therapy for ischemic stroke. The ability to measure the pO(2) in several sites provides important information that should help to optimize the design of hyperoxic therapies for stroke.

The effects of Efaproxyn (efaproxiral) on subcutaneous RIF-1 tumor oxygenation and enhancement of radiotherapy-mediated inhibition of tumor growth in mice.

Efaproxiral, an allosteric modifier of hemoglobin, reduces hemoglobin-oxygen binding affinity, facilitating oxygen release from hemoglobin, which is likely to increase tissue pO(2). The purpose of this study was to determine the effect of efaproxiral on tumor oxygenation and growth inhibition of RIF-1 tumors that received X radiation (4 Gy) plus oxygen breathing compared to radiation plus oxygen plus efaproxiral daily for 5 days. Two lithium phthalocyanine (LiPc) deposits were implanted in RIF-1 tumors in C3H mice for tumor pO(2) measurements using EPR oximetry. Efaproxiral significantly increased tumor oxygenation by 8.4 to 43.4 mmHg within 5 days, with maximum increases at 22-31 min after treatment. Oxygen breathing alone did not affect tumor pO(2). Radiation plus oxygen plus efaproxiral produced tumor growth inhibition throughout the treatment duration, and inhibition was significantly different from radiation plus oxygen from day 3 to day 5. The results of this study provide unambiguous quantitative information on the effectiveness of efaproxiral to consistently and reproducibly increase tumor oxygenation over the course of 5 days of treatment, modeling the clinical use of efaproxiral. Also, based on the tumor growth inhibition, the study shows the efaproxiral-enhanced tumor oxygenation was radiobiologically significant. This is the first study to demonstrate the ability of efaproxiral to increase tumor oxygenation and to increase the tumor growth inhibition of radiotherapy over 5 days of treatment.

Cerebral tissue oxygenation in reversible focal ischemia in rats: multi-site EPR oximetry measurements.

Multi-site electron paramagnetic resonance (EPR) oximetry was used in vivo to measure the partial pressure of oxygen (pO2) in reversible focal ischemia in rats. The cerebral tissue pO2 was measured simultaneously and continuously at two sites on the ischemic side and one on the normal side of the brain in the same animal prior to and at several time points after ischemia and reperfusion. The O2 at the three different sites in brain was stable over 30 min of baseline measurements. During the first 120 min of ischemia, statistically significant decreases in brain pO2 from baseline were consistently observed in the ischemic core and perifocal area. The mean values varied during the 120 min of ischemia. Reperfusion resulted in an immediate increase in PO2, but there were no significant differences between the sites over time. The result of this study seems promising for the study of ischemia and reperfusion. It appears that the technique can provide information on the PO2 under the experimental conditions needed for such a study. The levels of PO2 that occurred in these experiments are readily resolvable by multi-site EPR oximetry. In addition, the ability simultaneously to measure the pO2 in several sites provides important additional information that should help to differentiate between changes in the PO2 due toglobal or local mechanisms.

Modeling of the response of ptO2 in rat brain to changes in physiological parameters.

It is known that oxygen tension in tissue (ptO2) will change in response to an alteration of physiological parameters including: pCO2 in arterial blood, blood flow, capillary density, oxygen carrying capacity, and p50 of hemoglobin. We have used modeling to compute the change of PtO2 in response to changes of each physiological parameter and related these changes to experimental data. The oxygen distribution in a Krogh cylinder was computed assuming a linear decrease of hemoglobin saturation from the arterial to the venous end of the capillary. Parameters of the model were used to compute the baseline cerebral PtO2 expressed as the mean value of the PtO2 over the whole cylinder. These parameters were adjusted to derive PtO2 values close to those measured at the relevant experimental conditions. Then each desired parameter was varied to calculate the change in PtO2 related to this parameter. Effects of different factors on cerebral PtO2 were modeled and compared with experimental values obtained with various experimental interventions including: changing CBF, modifying p50 with the allosteric modifier RSR13, modification of capillary density, and hemoglobin content. An acceptable agreement of the computed and the experimental changes of the cerebral PtO2 was obtained for these experimental conditions.

Axial oxygen diffusion in the Krogh model: modifications to account for myocardial oxygen tension in isolated perfused rat hearts measured by EPR oximetry.

The cylindrical steady-state model developed by Krogh with Erlang has served as the basis of understanding oxygen supply in living tissue for over eighty years. Due to its simplicity and agreement with some observations, it has been extensively used and successfully extended to new fields, especially for situations such as drug diffusion, water transport, and ice formation in tissues. However, the applicability of the model to make even a qualitative prediction of the oxygen level of specific volumes of the tissue is still controversial. We recently have developed an approximate analytical solution of a steady-state diffusion equation for a Krogh cylinder, including oxygen concentration in the capillary. This model was used to explain our previous experimental data on myocardial pO2 in isolated perfused rat hearts measured by EPR oximetry. An acceptable agreement with the experimental data was obtained by assuming that a known limitation of the existing EPR methods--a tendency to over-weight low pO2 values--had resulted in an under-estimate of the pO2. These results are consistent with recent results of others, which stress the importance of taking into account the details of what is measured by various methods.

Cerebral PtO2, acute hypoxia, and volatile anesthetics in the rat brain.

We describe our results on the effect in rats of two commonly used, volatile anesthetics on cerebral tissue PO2 (PtO2) and other physiological parameters at FiO2 levels ranging from 0.35 to 0.1. The study was performed in 12 rats that had lithium phthalocyanine (LiPc) crystals implanted in the left cerebral cortex. FiO2 was maintained at 0.35 during surgical manipulation and baseline EPR measurements, after which time, each animal was exposed to varying levels of FiO2 (0.26, 0.21, 0.15, and 0.10) for 30 minutes at each level. No significant difference in PtO2 was observed between the isoflurane and halothane groups at any FiO2 level, and the cerebral arterial PO2 (PaO2) also was similar for both groups. However, the cerebral PtO2 under both isoflurane and halothane anesthesia was lower during hypoxia (FiO2 < or = 0.15) than under normoxia (FiO2 = 0.21) and there was a significant difference in mean arterial blood pressure (MABP) between isoflurane and halothane groups under both mild and severe hypoxia. The pH and cerebral arterial PCO2 (PaCO2) were similar for the halothane and isoflurane groups during normoxia (FiO2 = 0.21) and mild hypoxia (FiO2 = 0,15), but following severe hypoxia (FiO2 = 0.10), both parameters were lower in the halothane anesthetized animals. These results confirm that cerebral PO2 cannot be inferred directly from measurements of other parameters, indicating that methodology incorporating continuous direct measurement of brain oxygen will lead to a better understanding of cerebral oxygenation under anesthesia and hypoxia.

Simultaneous NIR-EPR spectroscopy of rat brain oxygenation.

Changes in cerebral oxygenation were simultaneously monitored by electric paramagnetic resonance (EPR) oximetry and near-infrared spectroscopy (NIRS). The tissue oxygen tension (t-pO2) was measured with an L-band (1.2 GHz) EPR spectrometer with an external loop resonator and the concentration of oxyhemoglobin [HbO2] and deoxyhemoglobin [Hb] were measured with a full-spectral NIRS system. Mean cerebral hemoglobin saturation (SmcO2) was calculated from the absolute [HbO2] and [Hb]. Six adult male rats were implanted with lithium phthalocyanine (LiPc) crystals into the left cerebral cortex. The change in oxygenation of the brain was induced by altering the inspired oxygen fraction (FiO2) in air from 0.30 at baseline to 0.0, 0.05, 0.10, and 0.15 for 1, 2, 5, and 5 minutes, respectively, followed by reoxygenation with an FiO2 = 0.30. Although both t-pO2 and SmcO2 values showed a decrease during reduced FiO2 followed by recovery on reoxygenation, it was found that SmcO2 recovered more rapidly than t-PO2 during the recovery phase. The recovery of t-pO2 is not only related to blood oxygenation, but also to delivery, consumption, and diffusion of oxygen into the tissue from the vascular system. Further studies will be required to determine the exact mechanisms for the delay between the recovery of SmcO2 and t-pO2.

pO2 and regional blood flow in a rabbit model of limb ischemia.

Oxygen tension (pO2) in muscles and regional blood flow were measured in a rabbit model of limb ischemia. pO2 was measured repetitively by EPR oximetry with EMS char in four different muscle groups in the same animals. Blood flow in the same muscles at several time points was measured using microspheres. A linear mixed effects model was developed to analyze the data on pO2 and blood flow. The results suggest that while under normal conditions pO2 in muscles does not depend significantly on blood flow, immediately after arterial occlusion pO2 correlates linearly with blood flow. Within two weeks of occlusion the pO2 is recovered to 45% of baseline. This study demonstrates, for the first time, the applicability of EPR oximetry in animals larger than rodents.

Tumor pO2 assessments in human xenograft tumors measured by EPR oximetry: location of paramagnetic materials.

Radioantibody immunotherapy (RAIT) is a promising treatment modality but the effectiveness of this targeted low dose radiation varies from tumor to tumor. Since RAIT is an oxygen dependent treatment, baseline pO2 or growth-induced changes in the microenvironment may alter treatment response. In this pilot work we monitored tumor pO2 in untreated human xenograft tumors growing s.c. in nude mice. These data will be used to plan a study of the relationship between the effectiveness of RAIT and tumor pO2. Growth or treatment-induced changes in the microenvironment may alter the tumor pO2 and thus affect the response to therapy but may also affect location and microenvironment of the particulate oxygen sensor. We monitored tumor pO2 during growth and also examined the tumor histological structure overall and in the region of the paramagnetic material in the tumor at the time of necropsy.

Electron paramagnetic resonance assessment of brain tissue oxygen tension in anesthetized rats.

UNLABELLED: The adequacy of cerebral tissue oxygenation (PtO(2)) is a central therapeutic end point in critically ill and anesthetized patients. Clinically, PtO(2) is currently measured indirectly, based on measurements of cerebrovascular oxygenation using near infrared spectroscopy and experimentally, using positron emission tomographic scanning. Recent developments in electron paramagnetic resonance (EPR) oximetry facilitate accurate, sensitive, and repeated measurements of PtO(2). EPR is similar to nuclear magnetic resonance but detects paramagnetic species. Because these species are not abundant in brain (or other tissues) in vivo, oxygen-responsive paramagnetic lithium phthalocyanine crystals implanted into the cerebral cortex are used for the measurement of oxygen. The line widths of the EPR spectra of these materials are linear functions of PtO(2). We used EPR oximetry in anesthetized rats to study the patterns of PtO(2) during exposure to various inhaled and injected general anesthetics and to varying levels of inspired oxygen. Rats anesthetized with 2.0 minimum alveolar anesthetic concentration isoflurane maintained the largest PtO(2) (38.0 +/- 4.5 mm Hg) and rats anesthetized with ketamine/xylazine had the smallest PtO(2) (3.5 +/- 0.3 mm Hg) at a fraction of inspired oxygen (FIO(2)) of 0.21, P < 0.05. The maximal PtO(2) achieved under ketamine/xylazine anesthesia with FIO(2) of 1.0 was 8.8 +/- 0.3 mm Hg, whereas PtO(2) measured during isoflurane anesthesia with FIO(2) of 1.0 was 56.3 +/- 1.7 mm Hg (P < 0.05). These data highlight the experimental utility of EPR in measuring PtO(2) during anesthesia and serve as a foundation for further study of PtO(2) in response to physiologic perturbations and therapeutic interventions directed at preventing cerebral ischemia. IMPLICATIONS: Using in vivo electron paramagnetic resonance oximetry, we studied the patterns of cerebral tissue oxygenation (PtO(2)) during exposure to various inhaled and injected general anesthetics, and to varying levels of inspired oxygen. These data show that inhaled anesthetics result in larger levels of PtO(2) in the brain than do several injectable anesthetics. The results highlight the experimental utility of electron paramagnetic resonance in measuring PtO(2) during anesthesia and serve as a foundation for further study of PtO(2) in response to physiologic perturbations and therapeutic interventions directed at preventing cerebral ischemia.