Cerebral oxygenation in awake rats during acclimation and deacclimation to hypoxia: an in vivo electron paramagnetic resonance study.

Exposure to high altitude or hypobaric hypoxia results in a series of metabolic, physiologic, and genetic changes that serve to acclimate the brain to hypoxia. Tissue Po(2) (Pto(2)) is a sensitive index of the balance between oxygen delivery and utilization and can be considered to represent the summation of such factors as cerebral blood flow, capillary density, hematocrit, arterial Po(2), and metabolic rate. As such, it can be used as a marker of the extent of acclimation. We developed a method using electron paramagnetic resonance (EPR) to measure Pto(2) in unanesthetized subjects with a chronically implanted sensor. EPR was used to measure rat cortical tissue Pto(2) in awake rats during acute hypoxia and over a time course of acclimation and deacclimation to hypobaric hypoxia. This was done to simulate the effects on brain Pto(2) of traveling to altitude for a limited period. Acute reduction of inspired O(2) to 10% caused a decline from 26.7 ± 2.2 to 13.0 ± 1.5 mmHg (mean ± SD). Addition of 10% CO(2) to animals breathing 10% O(2) returned Pto(2) to values measured while breathing 21% O(2,) indicating that hypercapnia can reverse the effects of acute hypoxia. Pto(2) in animals acclimated to 10% O(2) was similar to that measured preacclimation when breathing 21% O(2). Using a novel, individualized statistical model, it was shown that the T(1/2) of the Pto(2) response during exposure to chronic hypoxia was approximately 2 days. This indicates a capacity for rapid adaptation to hypoxia. When subjects were returned to normoxia, there was a transient hyperoxygenation, followed by a return to lower values with a T(1/2) of deacclimation of 1.5 to 3 days. These data indicate that exposure to hypoxia results in significant improvements in steady-state oxygenation for a given inspired O(2) and that both acclimation and deacclimation can occur within days.

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.

High spatial resolution multisite EPR oximetry of transient focal cerebral ischemia in the rat.

In vivo electron paramagnetic resonance (EPR) spectroscopy can provide direct noninvasive, continuous, and repeatable measurements of oxygen in tissues. High-spatial-resolution multisite (HSRMS) oximetry is an EPR technique that uses applied magnetic field gradients to extend this capability to multiple implanted probes within the sample and accurately to estimate their respective local pO(2) values. These capabilities are crucial in experiments in which pO(2) varies across space and time and in which information about these variations is needed to describe physiologic and pathophysiologic phenomena and evaluate their responses to interventions such as therapy. One important application is the investigation of transient focal ischemia in the rat brain and the effects of treatment with hyperoxygenation. We used HSRMS oximetry with overmodulation to measure brain tissue oxygenation in a rat stroke model using lithium phthalocyanine as the oxygen probe. Oxygen measurements were made in a small cohort of rats at four implant sites during ischemia and reperfusion after transient focal ischemia initiated by occlusion of the middle cerebral artery. These measurements demonstrate the capabilities of the HSRMS oximetry technique and set the stage for more extensive physiologic studies.

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.

Increased oxygenation of intracranial tumors by efaproxyn (efaproxiral), an allosteric hemoglobin modifier: In vivo EPR oximetry study.

PURPOSE: To determine quantitatively the changes in oxygenation of intracranial tumors induced by efaproxiral, an allosteric hemoglobin modifier. Efaproxiral reduces hemoglobin-oxygen binding affinity, which facilitates oxygen release from hemoglobin into surrounding tissues and potentially increases the pO(2) of the tumors. METHODS AND MATERIALS: The study was performed on 10 male Fisher 344 rats with 9L intracranial tumors. Electron paramagnetic resonance (EPR) oximetry was used to measure quantitatively the changes in the pO(2) in the tumors. Lithium phthalocyanine (LiPc) crystals were implanted in the tumors and in the normal brain tissue in the opposite hemispheres. We monitored the cerebral pO(2) starting 7 to 10 days after the tumor cells were implanted. NMR imaging determined the position and size of tumor in the brain. After an initial baseline EPR measurement, efaproxiral (150 mg/kg) was injected intravenously over 15 minutes, and measurements of tumor and normal brain oxygen tension were made alternately at 10-minute intervals for the next 60 minutes; the procedure was repeated for 6 consecutive days. RESULTS: Efaproxiral significantly increased the pO(2) of both the intracranial tumors and the normal brain tissue on all days. The maximum increase was reached at 52.9 to 59.7 minutes and 54.1 to 63.2 minutes after injection, respectively. The pO(2) returned to baseline values at 106 to 126.5 minutes after treatment. The maximum tumor and normal tissue pO(2) values achieved after efaproxiral treatment from Day 1 through Day 6 ranged from 139.7 to 197.7 mm Hg and 103.0 to 135.9 mm Hg, respectively. The maximum increase in tumor tissue pO(2) values from Day 2 to Day 5 was greater than the maximum increase in normal tissue pO(2). CONCLUSION: We obtained quantitative data on the timing and extent of efaproxiral-induced changes in the pO(2) of intracerebral 9L tumors. These results illustrate a unique and useful capability of in vivo EPR oximetry to obtain repeated noninvasive measurements of tumor oxygenation over a number of days. The information on the dynamics of tumor pO(2) after efaproxiral administration illustrates the ability of efaproxiral to increase intracranial tumor oxygenation.

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.

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.

Effect of RSR13, an allosteric hemoglobin modifier, on oxygenation in murine tumors: an in vivo electron paramagnetic resonance oximetry and bold MRI study.

PURPOSE: RSR13, an allosteric modifier of hemoglobin, reduces hemoglobin-oxygen binding affinity facilitating oxygen release from hemoglobin, resulting in increases in tissue pO(2). The purpose of this study was noninvasively to monitor the time course and effect of RSR13 on tumor oxygenation, directly using in vivo electron paramagnetic resonance (EPR oximetry), and indirectly using blood oxygen level dependent magnetic resonance imaging (BOLD MRI). METHODS AND MATERIALS: The study was performed in transplanted radiation-induced fibrosarcoma tumors (RIF-1) in 18 female C3H/HEJ mice, which had two lithium phthalocyanine (LiPc) deposits implanted in the tumor when the tumors reached about 200-600 mm(3). Baseline EPR measurements were made daily for 3 days. Then, for 6 consecutive days and after an initial baseline EPR measurement, RSR13 (150 mg/kg) or vehicle (same volume) was injected intraperitoneally, and measurements of intratumoral oxygen were made at 10-min intervals for the next 60 min. In each mouse, every third day, instead of EPR oximetry, BOLD MRI measurements were made for 60 min after administration of the RSR13. RESULTS: Based on EPR measurements, RSR13 produced statistically significant temporal increases in tumor pO(2) over the 60-min time course, which reached a maximum at 35-43 min postdose. The average time required to return to the baseline pO(2) was 70-85 min. The maximum increase in tumor tissue pO(2) values after RSR13 treatment from Day 1 to Day 5 (8.3-12.4 mm Hg) was greater than the maximum tumor tissue pO(2) value for Day 6 (4.7 mm Hg, p < 0.01). The maximum increase in pO(2) occurred on Day 2 (12.4 mm Hg) after RSR13 treatment. There was little change in R(2)*, indicating that the RSR13 had minimal detectable effects on total deoxyhemoglobin and hemoglobin-oxygen saturation. CONCLUSION: The extent of the increase in tumor pO(2) achieved by RSR13 would be expected to lead to a significant increase in the effectiveness of tumor radiotherapy. The lack of a change in the BOLD MRI signal suggests that the tumor physiology was largely unchanged by RSR13. These results illustrate a unique and useful capability of in vivo EPR oximetry and BOLD MRI to obtain repeated measurements of tumor oxygenation and physiology. The dynamics of tumor pO(2) after RSR13 administration may be useful for the design of clinical protocols using allosteric hemoglobin effectors.

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.

The dose-dependent effect of RSR13, a synthetic allosteric modifier of hemoglobin, on physiological parameters and brain tissue oxygenation in rats.

RSR13 is a synthetic allosteric modifier of hemoglobin that decreases the oxygen-binding affinity of hemoglobin, increasing the P50. As a result, tissue oxygen tension is expected to increase. Using the capabilities of in vivo EPR, we directly examined the effect of RSR13 on brain pO2 in rats and the relationship between any change in brain oxygenation and changes in physiological parameters, including blood gases. The brain pO2 and arterial blood paO2 were increased significantly (p < 0.005) following RSR13 administration. The peak increase of brain tissue pO2 was 8.8 +/- 1.2 mm Hg in the animals receiving 150 mg/kg RSR13 and 13 +/- 3 mm Hg in the animals receiving 300 mg/kg RSR13. There was no difference among groups in MBP, heart rate, paCO2, pH, or HCO3. These data indicate that in anesthetized rats, RSR13 dose-dependently increases brain pO2 without affecting other physiologic parameters. This capability is likely to be very useful in circumstances where the pO2 of the brain is compromised.

The effect of RSR13, a synthetic allosteric modifier of hemoglobin, on brain tissue pO2 (measured by EPR oximetry) following severe hemorrhagic shock in rats.

RSR13 is a synthetic allosteric modifier of hemoglobin that decreases the oxygen binding affinity of hemoglobin, potentially increasing oxygen availability to hypoxic tissues. Using in vivo EPR to directly measure cortical pO2, we examined whether RSR13 would improve brain tissue pO2 following severe hemorrhagic shock in rats. Hemorrhagic shock was induced by withdrawing blood (2.7-2.8 mL/100 g/15 min). Following a 30 min shock period, resuscitation was performed by infusion with Ringer lactate plus RSR13 (150 mg/kg) or saline (control). Following hemorrhage, brain pO2 decreased by about 14 mm Hg in both groups. Following crystalloid resuscitation brain pO2 remained depressed in the control group but returned to the pre-hemorrhage values in the rats that received RSR13. RSR13 immediately increased and maintained the paO2 while controls had a very gradual increase towards pre-hemorrhage values. There was no difference in the blood pressure or heart rate between groups. RSR13 may have useful applications to decrease the effects of acute hemorrhagic hypoxemia by increasing brain oxygenation.

The effects of anesthesia on cerebral tissue oxygen tension: use of EPR oximetry to make repeated measurements.

While very useful data can be obtained from measurements of pO2 within various compartments of the vascular system, such measurements do not necessarily provide accurate information on the pO2 in the brain. Anesthetics can significantly affect the tissue pO2 in the brain by several mechanisms involving both delivery and utilization. Electron Paramagnetic Resonance (EPR or ESR) oximetry has the potential for non-invasively carrying out repeated direct measurements of pO2 in tissues during the course of anesthesia. In this paper we describe the use of EPR oximetry for studying the influence of anesthesia on tissue pO2, and present illustrative results from experiments with five different anesthetics in rats. The results indicate that the tissue O2 can be measured directly using EPR oximetry, and data can be obtained non-invasively during the course of anesthesia.

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.

Changes in oxygenation of intracranial tumors with carbogen: a BOLD MRI and EPR oximetry study.

PURPOSE: To examine, using blood oxygen level dependent (BOLD) MRI and EPR oximetry, the changes in oxygenation of intracranial tumors induced by carbogen breathing. MATERIALS AND METHODS: The 9L and CNS-1 intracranial rat tumor models were imaged at 7T, before and during carbogen breathing, using a multi-echo gradient-echo (GE) sequence to map R(2)*. On a different group of 9L tumors, tissue pO(2) was measured using EPR oximetry with lithium phthalocyanine as the oxygen-sensitive material. RESULTS: The average decline in R(2)* with carbogen breathing was 13 +/- 1 s(-1) in the CNS-1 tumors and 29 +/- 4 s(-1) in the 9L tumor. The SI vs. TE decay curves indicate the presence of multiple components in the tumor. Tissue pO(2) in the two 9L tumors measured was 8.6 +/- 0.5 and 3.6 +/- 0.6 mmHg during air breathing, and rose to 20 +/- 7 and 16 +/- 4 mmHg (mean +/- SE) with carbogen breathing. Significant changes were observed by 10 minutes, but changes in pO(2) and R(2)* continued in some subjects over the entire 40 minutes. CONCLUSION: EPR results indicate that glial sarcomas may be radiobiologically hypoxic. Both EPR and BOLD data indicate that carbogen breathing increases brain tumor oxygenation. These data support the use of BOLD imaging to monitor changes in oxygenation in brain tumors.