Stalina Grinberg

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

Abstract Hou, H., Khan, N., Grinberg, O. Y., Yu, H., Grinberg, S. A., Lu, S., Demidenko, E., Steffen, R. P. and Swartz, H. M. The Effects of Efaproxyn™ (Efaproxiral) on Subcutaneous RIF-1 Tumor Oxygenation and Enhancement of Radiotherapy-Mediated Inhibition of Tumor Growth in Mice. Radiat. Res. 168, 218–225 (2007). Efaproxiral, an allosteric modifier of hemoglobin, reduces hemoglobin-oxygen binding affinity, facilitating oxygen release from hemoglobin, which is likely to increase tissue pO2. 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 pO2 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 pO2. 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.

Practical conditions and limitations for high-spatial-resolution multisite EPR oximetry

We have previously reported a high-spatial-resolution multisite electron paramagnetic resonance (EPR) oximetry method that is based on consecutive applications of magnetic field gradients with the same direction but different magnitudes. This method that could be called also two-gradient convolution EPR oximetry has no restrictions for the shape of solid paramagnetic materials implanted in tissue and is applicable for any particulate EPR oxygen-sensitive matieral with a Lorentzian line shape. To enhance the utilization of this method, a previously described algorithm was used to develop user-friendly Windows-based software. Practical conditions of application of the method were established using several different model systems. It has been shown that the spectral overlap from the adjacent sites can be neglected if the splitting between the corresponding lines exceeds the largest line width by at least a factor of 1.3. An additional requirement of the method is that the second field gradient should exceed by at least 30% the value of the first gradient. It was confirmed that the error in line width determination at L-band is proportional to the noise-to-signal ratio, and does not exceed 1% a noise-to-signal ratio of 0.1 in a typical in vivo experiment. We demonstrate that the line widths of up to 10 different sites can be determined.

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.

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.

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.

Axial Oxygen Diffusion in the Krogh Model

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.

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.

Impact of the Antimetastatic Drug Batimastat on Tumor Growth and PO2 Measured by Epr Oximetry in a Murine Mammary Adenocarcinoma

Patients treated for early breast cancer have only a 10–15% probability of local relapse (Fowble, 1991), but the likelihood of eventual recurrence with distant metastases can be much higher depending on the original stage of the cancer. It is from this systemic disease that patients die (Willner et al., 1997). Treatment of malignant tumors has relied heavily on the use of cytotoxic agents, targeted at the increased metabolic and proliferative activity of the cancer cell. An alternative strategy has been to develop low toxicity therapies that can be administered continuously over a period of several years to stabilize malignant disease, and it is in this category that treatment with matrix metallopro-teinase inhibitors (MMPIs) lie.