Oleg Y. Grinberg

The effects of ketamine–xylazine anesthesia on cerebral blood flow and oxygenation observed using nuclear magnetic resonance perfusion imaging and electron paramagnetic resonance oximetry

Ketamine-xylazine is a commonly used anesthetic for laboratory rats. Previous results showed that rats anesthetized with ketamine-xylazine can have a much lower cerebral partial pressure of oxygen (P(t)O(2)), compared to unanesthetized and isoflurane anesthetized rats. The underlying mechanisms for the P(t)O(2) reduction need to be elucidated. In this study, we measured regional cerebral blood flow (CBF) using nuclear magnetic resonance (NMR) perfusion imaging and cortical P(t)O(2) using electron paramagnetic resonance (EPR) oximetry in the forebrain of rats under isoflurane, ketamine, ketamine-xylazine and isoflurane-xylazine anesthesia. The results show that in ventilated rats ketamine at a dose of 50 mg/kg does not induce significant changes in CBF, compared to isoflurane. Ketamine-xylazine in combination causes 25-65% reductions in forebrain CBF in a region-dependent manner. Adding xylazine to isoflurane anesthesia results in similar regional reductions in CBF. EPR oximetry measurements show ketamine increases cortical P(t)O(2) while xylazine decreases cortical P(t)O(2). The xylazine induced reduction in CBF could explain the reduced brain oxygenation observed in ketamine-xylazine anesthetized rats.

Changes in Oxygenation of Intracranial Tumors With Carbogen: a BOLD MRI and EPR Oximetry Study

To examine, using blood oxygen level dependent (BOLD) MRI and EPR oximetry, the changes in oxygenation of intracranial tumors induced by carbogen breathing.

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.

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.

Noninvasive assessment of cerebral oxygenation during acclimation to hypobaric hypoxia.

Factors regulating cerebral tissue Po2 (PtO2) are complex. With the increased use of clinical PtO2 monitors, it has become important to elucidate these mechanisms. The authors are investigating a new methodology (electron paramagnetic resonance oximetry) for use in monitoring cerebral PtO2 in awake animals over time courses of weeks. The authors used this to study cerebral PtO2 in rats during chronic acclimation to hypoxia predicting that such acclimation would cause an increase in PtO2 because of increases that occur in capillary density and oxygen carrying capacity. The average PtO2 between 7 and 21 days was increased by 228% over controls.

Oxygen Consumption Rates and Oxygen Concentration in Molt-4 Cells and Their mtDNA Depleted (ρ0) Mutants

Respiratory deficient cell lines are being increasingly used to elucidate the role of mitochondria and to understand the pathophysiology of mitochondrial genetic disease. We have investigated the oxygen consumption rates and oxygen concentration in wild-type (WT) and mitochondrial DNA (mtDNA) depleted (rho(0)) Molt-4 cells. Wild-type Molt-4 cells have moderate oxygen consumption rates, which were significantly reduced in the rho(0) cells. PCMB (p-chloromercurobenzoate) inhibited the oxygen consumption rates in both WT and rho(0) cells, whereas potassium cyanide decreased the oxygen consumption rates only in WT Molt-4 cells. Menadione sodium bisulfite (MSB) increased the oxygen consumption rates in both cell lines, whereas CCCP (carbonyl cyanide m-chlorophenylhydrazone) stimulated the oxygen consumption rates only in WT Molt-4 cells. Superoxide radical adducts were observed in both WT and rho(0) cells when stimulated with MSB. The formation of this adduct was inhibited by PCMB but not by potassium cyanide. These results suggest that the reactive oxygen species (ROS) induced by MSB were at least in part produced via a mitochondrial independent pathway. An oxygen gradient between the extra- and intracellular compartments was observed in WT Molt-4 cells, which further increased when cells were stimulated by CCCP and MSB. The results are consistent with our earlier findings suggesting that such oxygen gradients may be a general phenomenon found in most or all cell systems under appropriate conditions.

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.

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 μM; air), the [O2] in the extracellular and phagosomal compartments was 180 and 141 μM, respectively. This was sufficient to maintain maximum superoxide production in these cells. When extracellular [O2] was reduced to 84 or 36 μM, the [O2] in phagosomes within the cells (31.7 and 7.7 μM, 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. J. Leukoc. Biol. 64: 78–84; 1998.

Effect of hyperventilation on brain tissue oxygenation and cerebrovenous PO2 in rats.

Previous studies have shown that cortical tissue oxygenation is impaired during hyperventilation. However, it is important to quantify the effect of hyperventilation on brain tissue PO(2) and cerebrovenous PO(2) simultaneously especially since cerebral venous oxygenation is often used to assess brain tissue oxygenation. The present study was designed to measure the sagittal sinus PO(2) (PvO(2)), brain tissue PO(2) in the thalamus (PtO(2)), and brain temperature (Bt) simultaneously during acute hyperventilation. Isoflurane-anesthetized rats were hyperventilated for 10 min during which time the arterial carbon dioxide tension (PaCO(2)) dropped from 40.3+4.9 mmHg to 23.5+2.8 mmHg. PtO(2) declined from 26.0+/-4.2 mmHg to 14.8+/-5.2 mmHg (P=0.004) while brain temperature decreased from 36.5+0.3 degrees C to 36.2+0.3 degrees C (P=0.02). However, PvO(2) and arterial blood pressure (BP) did not change during hyperventilation. The maintenance of PvO(2) when perfusion is thought to decline and PtO(2) decreases suggests that there may be a diffusion limitation, possibly due to selective perfusion. Therefore, cerebrovenous PO(2) may not give a good assessment of brain tissue oxygenation especially in conditions of acute hyperventilation, and deeper brain regions other than the cortex also show impaired tissue oxygenation following hyperventilation.

ELECTRON PARAMAGNETIC RESONANCE DOSIMETRY FOR A LARGE-SCALE RADIATION INCIDENT

Abstract With possibilities for radiation terrorism and intensified concerns about nuclear accidents since the recent Fukushima Daiichi event, the potential exposure of large numbers of individuals to radiation that could lead to acute clinical effects has become a major concern. For the medical community to cope with such an event and avoid overwhelming the medical care system, it is essential to identify not only individuals who have received clinically significant exposures and need medical intervention but also those who do not need treatment. The ability of electron paramagnetic resonance to measure radiation-induced paramagnetic species, which persist in certain tissues (e.g., teeth, fingernails, toenails, bone, and hair), has led to this technique becoming a prominent method for screening significantly exposed individuals. Although the technical requirements needed to develop this method for effective application in a radiation event are daunting, remarkable progress has been made. In collaboration with General Electric and through funding committed by the Biomedical Advanced Research and Development Authority, electron paramagnetic resonance tooth dosimetry of the upper incisors is being developed to become a Food and Drug Administration-approved and manufacturable device designed to carry out triage for a threshold dose of 2 Gy. Significant progress has also been made in the development of electron paramagnetic resonance nail dosimetry based on measurements of nails in situ under point-of-care conditions, and in the near future this may become a second field-ready technique. Based on recent progress in measurements of nail clippings, it is anticipated that this technique may be implementable at remotely located laboratories to provide additional information when the measurements of dose on-site need to be supplemented. The authors conclude that electron paramagnetic resonance dosimetry is likely to be a useful part of triage for a large-scale radiation incident.