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J Appl Physiol 90: 1887-1899, 2001;
8750-7587/01 $5.00
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Vol. 90, Issue 5, 1887-1899, May 2001

Oxygen measurements in brain stem slices exposed to normobaric hyperoxia and hyperbaric oxygen

Daniel K. Mulkey1, Richard A. Henderson III2, James E. Olson1,3, Robert W. Putnam1, and Jay B. Dean1

1 Department of Physiology and Biophysics, Environmental and Hyperbaric Cell Biology Facility, 2 Department of Community Health, and 3 Department of Emergency Medicine, College of Science and Mathematics, Wright State University School of Medicine, Dayton, Ohio 45435


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

We previously reported (J Appl Physiol 89: 807-822, 2000) that <= 10 min of hyperbaric oxygen (HBO2; <= 2,468 Torr) stimulates solitary complex neurons. To better define the hyperoxic stimulus, we measured PO2 in the solitary complex of 300-µm-thick rat medullary slices, using polarographic carbon fiber microelectrodes, during perfusion with media having PO2 values ranging from 156 to 2,468 Torr. Under control conditions, slices equilibrated with 95% O2 at barometric pressure of 1 atmospheres absolute had minimum PO2 values at their centers (291 ± 20 Torr) that were ~10-fold greater than PO2 values measured in the intact central nervous system (10-34 Torr). During HBO2, PO2 increased at the center of the slice from 616 ± 16 to 1,517 ± 15 Torr. Tissue oxygen consumption tended to decrease at medium PO2 >=  1,675 Torr to levels not different from values measured at PO2 found in all media in metabolically poisoned slices (2-deoxy-D-glucose and antimycin A). We conclude that control medium used in most brain slice studies is hyperoxic at normobaric pressure. During HBO2, slice PO2 increases to levels that appear to reduce metabolism.

solitary complex; polarographic oxygen measurements; metabolism; reactive oxygen species; central nervous system oxygen toxicity


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

THE IN VITRO BRAIN SLICE PREPARATION has been used for more then 40 years to study neuronal excitability because it allows considerable control of the neuronal environment while retaining local neuronal circuitry (42). Brain slices are removed from blood supply and receive oxygen solely by diffusion from the nutrient medium that bathes the tissue. To ensure adequate oxygenation of cells lying deep to the slice surface, most brain slice studies use 95% O2 to set the PO2 in the control medium. At a barometric pressure (PB) of 1 atmosphere absolute (ATA),1 this produces a PO2 in the nutrient medium of ~722 Torr. Under this condition, brain slices remain viable for up to 8 h, based on electrophysiological criteria (12). Consequently, it has generally been assumed that PO2 in the core of a submerged slice is adequate (40).

Several studies have reported tissue PO2 values in brain slices measured with polarographic microelectrodes (3-5, 19-21, 28, 50, 56). These experiments were done to determine the slice thickness that optimized slice viability, as measured by extracellularly recorded field potentials, while ensuring that an anoxic tissue core was avoided (19), or, alternatively, to identify the level of oxygenation in the slice so that electrophysiological data could be correlated with tissue PO2 during hypoxia (28, 50, 56) and hyperoxia (3-5, 56). Because PO2 at any depth in a slice is determined by PO2 of the perfusate, oxygen diffusion distance into the slice, and oxygen consumption (VO2), tissue PO2 measurements were also used to determine VO2 (20, 21). Results from these studies varied, however, because of differences in slice thickness, central nervous system (CNS) regions, animal age, and orientation of slice surfaces relative to the supporting structure (nylon mesh vs. solid Plexiglas support) and the fluid-gas interface (interface slice vs. submerged slice) (4, 19, 28). Nevertheless, under control conditions, 300- to 450-µm-thick brain slices had minimum PO2 values that were consistently higher (19, 20, 28, 50) than those measured in the intact CNS (9, 23, 24, 27, 51, 61).

We studied the effects of hyperoxia, reactive oxygen species (ROS), and antioxidants on the electrophysiology of neurons in the solitary complex (12, 44-46), an important cardiorespiratory control center in the caudodorsal medulla oblongata (14, 18). The challenge of studying hyperoxia in rat brain slices, however, is that the standard control PO2 of the medium used in this preparation is already hyperoxic at normobaric pressure (PB of ~1 ATA). Increasing tissue PO2 further requires increasing the PB of the slice and nutrient media together with a gas mixture containing a high fractional concentration of O2 (Fo2). Our initial findings, under conditions of <= 10 min of hyperbaric oxygen (HBO2; i.e., Fo2 = 95-98% O2 at PB of 2.4-3.3 ATA), indicate a subpopulation of neurons in the solitary complex that are depolarized, exhibit increased firing rate, and, typically, have decreased membrane conductance (12, 45-46). However, these neuronal responses to HBO2 may be blunted because slices recorded under control conditions are already hyperoxic. Moreover, we are concerned that under control conditions neuronal activity is altered by the high PO2 (3, 5, 56) and increased exposure to ROS (26, 33, 54, 55). Previous electrophysiological studies in brain slices found that neuronal activity recorded in medium equilibrated with 21% O2 is different from neuronal activity recorded in medium equilibrated with 95% O2 (3, 5, 56). Investigators have proposed that these differences in excitability seen with 21% oxygen are not due to hypoxia, which is typically studied using 10-15% O2 (35), but rather to normobaric hyperoxia with 95% oxygen (3, 5, 56), possibly by an increased production of ROS (26).

The goal of the present study was to measure PO2 in both perfusion media and the solitary complex in slices prepared from weaned and adult rats under the same experimental conditions used in our electrophysiology studies. In doing so, we will be able to correlate changes in neuronal excitability recorded during HBO2 with known changes in tissue PO2. Moreover, we wanted to determine the degree of hyperoxia within our brain slice model under control conditions at normobaric pressure (PB of ~1 ATA). We hypothesize that tissue PO2 will decrease in the solitary complex under control conditions (95% O2 at PB of ~1 ATA) with increasing tissue depth; however, tissue PO2 at the center of the slice will remain hyperoxic compared with tissue PO2 in the intact CNS. We also hypothesize that slice PO2 will increase significantly during HBO2. Finally, we hypothesize that tissue VO2 will decrease during HBO2 because it has previously been shown that increasing PO2 of the perfusate from 150 to 600 Torr at normobaric pressure increased VO2 (4), whereas HBO2 reduced cellular VO2 (1, 11). A preliminary report of these data was previously published (47).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

Pressure Terminology

The partial pressure of oxygen is the product of PB and Fo2. When varying PB, it is important to define the PO2 of the perfusate and tissue slice relative to PB, especially when PB and PO2 are independently manipulated (12). "Normobaric pressure" refers to ambient pressure measured in our laboratory with a mercury barometer; this was slightly less than normal PB at sea level (~1 ATA or ~760 Torr), typically ranging from 738 to 752 Torr.2 "Hyperbaric pressure" refers to ambient pressure inside the hyperbaric chamber that is greater than 1 ATA. "Normoxia" refers to slice PO2 values that approximate values measured in vivo from rats that breathed air (20-21% O2) at normobaric pressure, i.e., CNS tissue PO2 of ~10-34 Torr (Table 1). "Normobaric hyperoxia" refers to slice PO2 values greater than those measured in vivo from rats breathing air at PB ~1 ATA, i.e., >34 Torr (Table 1). Conventional brain slice control medium, including the artificial cerebral spinal fluid (aCSF) used in this study, was equilibrated with 95% O2-5% CO2 at normobaric pressure; thus, under control conditions, the slice was exposed to hyperoxic medium; in this study, control medium PO2 values were ~708 Torr. "HBO2" in this report describes any perfusate with PO2 of >760 Torr or 1 ATA. In the present study, slices were exposed to three different HBO2 values depending on PB, designated here in ATA after the dash (e.g., HBO2-2 signifies hyperoxic medium at a PB of 2 ATA). The HBO2 PO2 values used were 1,200, 1,675, and 2,468 Torr. In this way, tissue can be exposed to hyperoxia at both normobaric pressure and hyperbaric pressure.

                              
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  		Table 1.   Oxygen electrode measurements of PO2 in CNS tissue, cerebrospinal fluid, and arterial blood in anesthetized rats and humans breathing air, O2, and CO2 gas mixtures at normobaric pressure and hyperbaric pressure

Brain Slices and Control Media

Slices were prepared from weaned and adult Sprague-Dawley rats as previously described (12). Anesthesia was not used because of the depressant actions these agents have on neurons (49) and their reported antagonistic interactions with elevated PB (31, 53, 59). After decapitation, the brain stem was isolated and submerged in ice-cold (4-6°C) aCSF of the following composition (in mM): 125 NaCl, 5 KCl, 1.3 MgSO4, 26 NaHCO3, 1.24 KH2PO4, 2.4 CaCl2, 10 glucose at 300 mosM, pH of ~7.45 and PO2 of ~708 Torr after equilibration with a 95% O2-5% CO2 gas mixture at PB of ~1 ATA. Hyperoxia (22) and HBO2 (38, 57) both affect central respiratory control; therefore, we chose to study the effects of oxygen on a part of the brain involved in respiratory control, namely, the solitary complex. Transverse slices were cut at 300 µm starting from the obex and proceeding rostrally through the medulla oblongata. Slices were incubated in control medium at ~25°C for at least 1 h before one was selected and transferred to a tissue chamber inside the hyperbaric chamber (12). Brain slices typically remained viable for electrophysiological studies under these conditions for up to 8 h (12).

Hyperbaric Chamber

A detailed description of the hyperbaric chamber, sample cylinders, and tissue chamber are given elsewhere (13). Briefly, the hyperbaric chamber has a maximum working pressure of 65 ATA. Within the hyperbaric chamber, tissue was submerged in aCSF that was delivered at a rate of 2 ml/min using one of two high-pressure liquid chromatography (HPLC) pumps. The brain slice rested on a fine-mesh nylon grid and was stabilized by placing a large-mesh nylon grid over the top surface (Fig. 1). Temperature of the tissue bath and air above the preparation was regulated at 37 ± 0.3°C by a servo-controlled two-channel temperature controller. The tissue chamber and electronic microdrive, which was used to maneuver the PO2 electrode by remote control, and various other equipment items were positioned on a retractable sled for easy access when the hyperbaric chamber was opened. Once the equipment sled was pushed in and the chamber door was sealed, the tissue slice and oxygen electrode were visualized using an externally mounted stereoscope positioned over a window in the top of the chamber. As in previous studies (12, 16, 44-48, 59), pure helium was used to hydrostatically compress the tissue bath and, hence, the brain slice. Helium is inert and of low solubility in aqueous and lipid media (2), thus helium has no partial pressure effect [e.g., at PB >=  3 ATA, nitrogen can act as an anesthetic (31)] over the range of ambient pressures used in our study. Before compression, room air was purged from the chamber atmosphere and replaced with 100% helium; the chamber was then compressed at a rate of 2 atm/min.


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  		Fig. 1.   Illustration of the brain slice orientation in relation to the flow of fresh oxygenated artificial cerebral spinal fluid (aCSF; not drawn to scale). Fresh aCSF enters the tissue chamber from the bottom and flows around and over the slice while a filter paper wick (not shown) draws it away from the surface.

Test Conditions

Equilibrating aCSF at PB of ~1 ATA and 37°C with 95% N2 or 95% air (balance CO2) resulted in media with PO2 values of ~0 and ~156 Torr, respectively. Hyperbaric oxygenated medium (i.e., medium that has been supersaturated with oxygen) was made by equilibrating aCSF with 98.3% O2 at 1.6, 2.2, or 3.3 ATA in separate high-pressure sample cylinders (1-liter volume) to produce corresponding medium PO2 values of ~1,200, 1,657, and 2,468 Torr. No attempt was made to keep PCO2 constant at PO2 of 1,200 and 1,657 Torr; however, it is possible to do so by reducing the fractional concentration of CO2 with increased PB (12). A pressure differential of 0.13-0.6 ATA was used to deliver hyperoxic aCSF to the tissue chamber. A high-pressure solenoid valve (General Valve, Fairfield, NJ) was used to rapidly select between control and hyperoxic medium such that perfusate flow rate was not significantly disrupted.

Oxygen Measurements

Oxygen was measured using a carbon fiber needle electrode (tip outer diameter of ~10 µm), previously described by Jiang et al. (28). Electrodes were constructed by sealing an 8-µm-diameter carbon fiber (Alfa Aesar, Ward Hill, MA) at the tip of a glass pipette (MTW150-6, World Precision Instruments, Sarasota, FL) using Duco cement. The other end of the fiber was attached to a copper wire using graphite conductive adhesive (Alpha Aesar) and connected to the input of a polarographic oxygen amplifier (A-M Systems, model 1900). A -0.6-V potential was imposed between the oxygen electrode and a low-resistance (<1.0 MOmega ) Ag/AgCl reference that was in contact with the tissue bath via a potassium gluconate agar bridge (12). Oxygen electrodes were typically calibrated before and after each profile in aCSF equilibrated with 21 and 95% oxygen; only electrodes that showed a 3.5- to 4.5-fold current difference between media were used (typical slope varied between 5-10 pA/Torr).

Oxygen profiles were made by lowering the oxygen electrode in 50-µm steps perpendicular to the tissue surface. Recording depth in tissue was approximated two ways: 1) surface depth was determined by moving the electrode down in small steps and then moving it laterally until the tip of the electrode touched the tissue, as seen by a bowing of the electrode shank during lateral movement; and 2) core tissue depth was identified as the depth at which PO2 was minimum (4, 19).

Absolute PO2 values presented here were obtained directly from continual PO2 recordings stored as AxoScope records (Axon Instruments, Foster City, CA) and/or on magnetic tape (Vetter PCM recorder model 400, Rebersburg, PA). Approximately one-half of the electrodes used developed drift after more than ~0.5 h in the slice, probably as the result of tissue debris on the tip (10), and resulted in an offset of 91 ± 56 Torr. If an offset developed in the measured PO2, the presented values were the sum of both the measured PO2 plus any offset.

Metabolic Block Media

To minimize tissue VO2, all metabolizable glucose in the aCSF was replaced with 1 mM 2-deoxy-D-glucose (2DG; Sigma Chemical, St. Louis, MO). Oxygen measurements also were made in 2DG medium supplemented with 9.1 nM antimycin A (Sigma Chemical). Antimycin A, an antibiotic that blocks electron flow from cytochrome b to c1 (29), was added to the 2DG aCSF to block metabolism of substrates other then glucose (e.g., lactate).

Data Collection and Analysis

Data were collected and analyzed using a 486 PC and the AxoScope 7.0, Origin 5.0, and Mathematica software packages. Statistical significance was determined at P < 0.05 by one-way ANOVA and multiple comparison tests (Tukey's or Newman-Keuls) or Student's t-test. Linear regressions were also compared using analysis of covariance. Data are presented as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

PO2 Measurements in the Tissue Bath

PO2 electrode calibrations. Figure 2A shows five superimposed PO2 traces made with the oxygen electrode submerged deep into the tissue bath in the absence of a brain slice. The recordings were initiated in control aCSF with a PO2 of 708 Torr. The perfusate source was then switched to aCSF with PO2 values of 0, 156, 1,200, 1,657, and 2,468 Torr to produce normobaric anoxia, 21% oxygen, HBO2-1 (PB of 1 ATA), HBO2-2 (PB of 2 ATA), and HBO2-3 (PB of 3 ATA), respectively. During HBO2-1, PO2 recordings were less stable because, as the pressure differential between the medium and tissue chamber approached 2:1, small oxygen bubbles would form in the aCSF inflow line and tissue bath, which disrupted the perfusate flow rate and aCSF meniscus in the tissue chamber. When the control medium was switched to one of the test media, a short delay in the electrode response was observed due to the dead space between the medium reservoirs and tissue chamber. Figure 2B shows that the polarographic electrode current measured at the plateau phase of each curve in Fig. 2A was linearly proportional to medium PO2 at both normobaric and hyperbaric pressure over a range of PO2 values from 0 to 2,468 Torr.


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  		Fig. 2.   A: continuous traces of oxygen partial pressure measured deep in the tissue bath while switching aCSF from control (PO2 ~710 Torr) to aCSF with PO2 values of 0, 156, 1,200, 1,657, and 2,468 Torr. All recordings were made at 37°C and at constant barometric pressure (PB). Bottom 3 traces (anoxia, 21% oxygen, and HBO2-1, where HBO2 signifies hyperbaric O2) were recorded at 1 atmospheres absolute (ATA), whereas the upper two traces (HBO2-2 and HBO2-3) were recorded while the chamber pressure was held at 2 and 3 ATA, respectively. B: triplicate individual current values measured during the plateau phase of each medium PO2, excluding HBO2-1. Carbon fiber electrode produced a current that was linearly proportional to PO2 from 0 to 2,468 Torr (9.6 pA/Torr, r2 = 0.97, n = 15).

Gas-liquid oxygen diffusion gradient. At an interface between gas and liquid media with dissimilar oxygen tensions, oxygen will diffuse down its chemical gradient. In our submerged slice preparation, oxygenated aCSF was in contact with an anoxic gaseous atmosphere (100% helium). Consequently, PO2 was measured as a function of aCSF depth into the tissue bath to determine the extent to which medium PO2 dropped as a result of diffusion into the chamber atmosphere. The PO2 measurements shown in Fig. 3 were made in the tissue bath without a brain slice present. The recordings were initiated at the tissue bath surface in aCSF, with PO2 values of ~708, 1,657, or 2,468 Torr. The electrode was then moved through the aCSF in 50-µm steps until the recorded PO2 reached a stable plateau. These measurements show that bath PO2 increased at depths into medium between 0 and ~450 µm, thus signifying the presence of an oxygen diffusion layer that was probably due to a loss of oxygen from the aCSF to the chamber atmosphere. The relative oxygen gradient, expressed as a percentage of the maximum PO2 at 450 µm, at each medium PO2, was similar. For instance, the steady-state PO2 at depths of 100, 250, or 450 µm, were ~20, 50, or 100%, respectively, of the maximum PO2. The independence of the relative oxygen diffusion gradient from medium PO2 may result from the configuration of our perfusion system. Fresh oxygenated aCSF is delivered to the tissue chamber from the bottom where it flows up toward the surface. We assumed that PO2 of the chamber atmosphere remained negligible since the hyperbaric chamber volume was considerably larger (72 liters) than the tissue chamber volume (~ 5 ml) and the frequent flushing of the hyperbaric chamber atmosphere with fresh helium gas further minimized any PO2 buildup.


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  		Fig. 3.   An oxygen diffusion gradient was measured under control and hyperoxic conditions in the absence of a brain slice. Oxygen measurements were initiated while the oxygen electrode was positioned at the surface of the aCSF. Then, moving in 50-µm increments, the electrode was driven into the aCSF, which flowed into the ~5-ml chamber at a rate of 2 ml/min. Medium PO2 was lowest at the interface where O2 diffused into the gas phase of the overlying atmosphere. Medium PO2 increased with depth reaching a maximum value at a depth of ~450 µm for each medium. The results were similar for 2 additional trials using HBO2.

PO2 Measurements in the Slice

Regular aCSF. A total of 38 PO2 profiles were made in 300-µm-thick brain slices perfused with aCSF having PO2 values that ranged from 156 to 2,468 Torr. Examples of individual profiles made at 708, 1,657, and 2,468 Torr are shown in Fig. 4. The brain slice was positioned >= 500 µm from the gas-liquid interface. The recordings began while the electrode was positioned 200 µm above the tissue surface. Although the distance of the initial recording position from the surface of the bath was not measured directly, it was estimated to be 250-350 µm, based on the gas-liquid oxygen diffusion gradient (Fig. 3), assuming that the slice does not influence oxygen diffusion into the chamber atmosphere. In contrast to Fig. 3, PO2 decreased in the aCSF as the oxygen electrode moved deeper into the bath toward the surface of the submerged tissue slice in Fig. 4, undoubtedly due to VO2 by the slice. Once the oxygen electrode was in the slice, PO2 decreased further to a minimum at the slice core (~150 µm), after which tissue PO2 increased as the oxygen electrode approached the lower surface of the slice. Likewise, PO2 in the bath increased as the electrode moved away from the lower surface of the slice. This indicated that an oxygen diffusion layer in the aCSF (~200 µm thick) was present around each surface of the tissue, with a magnitude roughly equivalent to the oxygen diffusion gradient in the first 100 µm of tissue. Figure 5 summarizes the dynamics of oxygen diffusion in our submerged slice preparation and further illustrates the significant effect that these oxygen diffusion gradients have on setting PO2 in the aCSF and slice.


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  		Fig. 4.   Continual PO2 measurements were made through 300-µm-thick brain slices equilibrated with control (PO2 ~ 708 Torr) or hyperoxic (HBO2-2 and HBO2-3) aCSF. For the top 2 traces, the perfusate was switched to hyperoxic medium and allowed to establish a new stable PO2 before the electrode was moved toward the tissue in 50-µm steps. Profiles were initiated 200 µm above the surface of slice. The designation "ST" signifies when the electrode was closest to the top surface of the slice; "SB" signifies when the electrode was closest to the slice bottom. With each step, the corresponding change in PO2 was allowed ~1 min to stabilize before the next step. The depth scale under the PO2 traces corresponds to the control PO2 profile. Notice that PO2 declined above the slice and increased below the slice, thus indicating the presence of a diffusion layer at each surface of the slice.



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  		Fig. 5.   PO2 traces were plotted as a function of aCSF and slice depth. Traces of PO2 made during control (708 Torr) and HBO2-3 (2,468 Torr) conditions show bath PO2 increasing with distance (50-µm steps) from the aCSF meniscus. As the electrode approached the surface of the slice, PO2 began decreasing. *Not a continuous recording. Arrows at the He gas-aCSF and slice-aCSF interfaces indicate the direction of oxygen diffusion. Measured PO2 reached a relative minimum at the slice core, after which PO2 increased as the electrode passed through and away from the bottom of the brain slice. Typically, PO2 recordings were terminated ~200 µm past the bottom of the slice.

Tissue PO2 measured at the surface (0 µm) of the slice and at a maximum depth of 150 µm were averaged to estimate mean tissue PO2 and plotted against medium PO2 in Fig. 6. Table 2 summarizes mean tissue PO2 plotted in Fig. 6 as well as values measured at a depth of 50 and 100 µm into tissue. Increasing PO2 of the medium from 156 to 2,468 Torr increased tissue PO2 measured at 0 and 150 µm depths proportionally; slopes of regression lines were 0.65 ± 0.02 (r2 = 0.998, n = 38) and 0.66 ± 0.04 (r2 = 0.997, n = 38) at the surface and core, respectively. However, mean tissue PO2 measured at 0 and 150 µm were significantly different at medium PO2 values of 156, 708, and 1,200 Torr. As medium PO2 increased beyond 1,200 Torr, there was no statistical difference in mean PO2 measured at the tissue surface and tissue core. These results suggest slice VO2 may have decreased during exposure to the higher levels of HBO2 (see Metabolically Poisoned Tissue below).


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  		Fig. 6.   Mean PO2 (n = 3-13, see Table 1) measured in regular aCSF at the slice surface (0 µm) and at the center of the slice (150 µm) were plotted against media PO2. Oxygen tension measured at 0 µm differed significantly (*P < 0.05) from values measured at 150 µm at PO2<= 1,200 Torr. At a constant PO2 of ~708 Torr, slice PO2 at 150 µm was measured in slices compressed with helium to PB of 1, 2, and 3 ATA, and these mean PO2 values (n = 3) were plotted as the dashed line. At ~708 Torr, PO2 measured at 2 and 3 ATA were not significantly different from PO2 measured at 1 ATA.


                              
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  		Table 2.   Mean tissue PO2 in regular aCSF and in aCSF with 2DG and antimycin A

In our electrophysiological studies of how HBO2 affects neuronal excitability (12, 45, 46), it was important to differentiate the effects of pressure per se (i.e., hyperbaric helium) from those of high PO2; thus tissue PO2 measurements were made in tissue equilibrated with a constant control level of PO2 (~708 Torr) at PB of 1, 2, and 3 ATA. The hyperbaric chamber was compressed with 100% helium and allowed 2-5 min for equilibration before tissue PO2 was measured at a depth of 150 µm. Results from these measurements are plotted in Fig. 6. Tissue PO2 at 2 and 3 ATA were not significantly different from PO2 measured at 1 ATA, and the slope of the regression line, 0.04 ± 0.04 (r2 = 0.772, n = 3), was not significantly different from zero. These results indicate that the effect of pressure per se can be differentiated from increased oxygen tension. It also indicates that 2 and 3 ATA of pressure do not affect oxygen diffusion or utilization (12, 47).

Metabolically poisoned tissue. A series of PO2 profiles were made in slices incubated at medium PO2 values ranging from 156 to 2,468 Torr in 2DG aCSF or 2DG plus antimycin A. This was done to determine how VO2 affects slice PO2 (i.e., the magnitude of PO2 profiles between the slice surface and the core of the slice). The difference in PO2 measured at the slice surface (0 µm) to its core (150 µm) was defined as delta PO2 (Delta PO2). By comparing the Delta PO2 measured in metabolically active slices at different medium PO2 values with the same measurements made in metabolically poisoned slices, the PO2 dependence of VO2 could be determined. Mean slice PO2 measured at 150 µm in slices perfused with 2DG and 2DG plus antimycin A medium was linearly related to medium PO2. No significant difference existed between the slopes of regression lines for each data set; therefore, the 2DG data and antimycin A supplemented 2DG data were pooled. The slope of pooled data vs. medium PO2 was 0.74 ± 0.03 (r2 = 0.997, n = 33).

Figure 7A shows superimposed PO2 profiles, measured at 708 Torr, in regular and a metabolically poisoned brain slice. For comparison, mean PO2 values measured at depths of 0, 50, 100, and 150 µm in metabolically poisoned slices are given in Table 2. In this example, Delta PO2 was smaller in metabolically poisoned tissue (Delta PO2 = 66 Torr) compared with metabolically active tissue (Delta PO2 = 116 Torr). We considered the difference in Delta PO2 between metabolically active tissue and 2DG tissue to be proportional to VO2. Consequently, Delta PO2 in nonpoisoned tissue was used as an indirect measure of VO2 to gain insight as to how HBO2 affects VO2. Mean Delta PO2 values were measured in metabolically active and inactive tissue and plotted against medium PO2 in Fig. 7B. Mean Delta PO2 values measured in metabolically poisoned slices did not vary significantly over the entire range of medium PO2 values studied. This indicated that the effects of PO2 on nonmetabolic forms of oxygen utilization (e.g., formation of ROS) were negligible in this comparison. However, Delta PO2 measured in metabolically active slices at 708 Torr was significantly greater than Delta PO2 measured in metabolically poisoned slices; however, as medium PO2 increased from 708 to 2,468 Torr, Delta PO2 values measured in metabolically active slices got smaller and more closely resembled profiles in metabolically poisoned slices, thus suggesting VO2 was reduced during exposure to HBO2.


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  		Fig. 7.   A: 2 superimposed PO2 profiles measured at ~708 Torr in a metabolically active and metabolically inactive slice. The difference in PO2 from the slice surface (0 µm) to its core (150 µm) was defined as delta (Delta ) PO2. B: Delta PO2 measured in metabolically active tissue at a PO2 of ~708 Torr was significantly greater (P < 0.05) than Delta PO2 in metabolically poisoned tissue at all PO2 tested. During HBO2, the Delta PO2 values measured in metabolically active brain slices were reduced to values that more closely resembled Delta PO2 in metabolically poisoned slices. *PO2 measured in metabolically active slices at which the Delta PO2 was significantly (P < 0.05) smaller than Delta PO2 measured at ~708 Torr. These results suggest that oxygen consumption is reduced by HBO2.

The oxygen diffusion coefficient (D) and VO2 were calculated to better describe the dynamics of oxygen in our preparation over a broad range of PO2 values as well as to quantify, if only by approximation, the relationship between VO2 and tissue PO2 (see APPENDIX for details regarding the calculations of D and VO2). During measurements of oxygen at a constant depth of 150 µm in a metabolically poisoned brain slice, D was determined by measuring the change in PO2 over time when switching between two media with different nonzero PO2 values (8, 17), in this case 708 and 156 Torr. With the assumption that all forms of oxygen utilization were constant, D was estimated to be 1.3 × 10-6 cm2/s. Our estimated D was smaller by about one-tenth than the D calculated from PO2 measurements in 1,000-µm-thick slices of cat cortex equilibrated with a similar initial PO2, 1.54 × 10-5 cm2/s (21).

VO2 rate can be calculated from tissue oxygen profiles using Fick's second law of diffusion (20). With the use of boundary conditions of PO2 at the surface of the slice (defined as P0) and at the bottom of the slice [defined as PL; thickness of the slice (L) = 300 µm], our slice PO2 profiles at each medium PO2 were fitted to the parabolic equation (20)
P<SC>o</SC><SUB><IT>2</IT></SUB><IT>=</IT>a<IT>X<SUP>2</SUP>−</IT><FENCE>a<IT>L+</IT><FR><NU>P<SUB><IT>0</IT></SUB><IT>−</IT>P<SUB><IT>L</IT></SUB></NU><DE><IT>L</IT></DE></FR></FENCE><IT>x+</IT>P<SUB><IT>0</IT></SUB>
where PO2 is oxygen tension measured at depth X in a brain slice, a = VO2/2DS, and S is the estimated solubility coefficient of oxygen in cat brain (1.89 × 10-5 ml O2 · cm-3 · Torr-1) (21). Using our estimated D (1.3 × 10-6 cm2/s), we calculated the VO2 (mean ± SE ml O2 · cm-3 · min-1) at each medium PO2 to be 1.4 ± 0.17 × 10-3 at 156 Torr, 1.8 ± 0.2 × 10-3 at 708 Torr, 1.9 ± 0.32 × 10-3 at 1,200 Torr, 1.7 ± 0.75 × 10-3 at 1,657 Torr, and 9.3 ± 1.3 × 10-4 at 2,468 Torr. Although VO2 at 1,200 and 1,657 Torr were not less than VO2 at 708 Torr (i.e., VO2 and Delta PO2 were not well matched), a trend of decreasing VO2 was evident at 2,468 Torr. These results suggest VO2 was dependent on PO2 during hyperoxia; therefore, we incorporated this assumption into our calculation of VO2 (see APPENDIX) and estimated VO2 at a constant depth by measuring the rate of change in PO2 measured in a metabolically active slice exposed to different aCSF PO2 values (8). Oxygen measurements were made at a constant depth of 150 µm in a brain slice while media PO2 changed from ~708 to ~156 Torr or from ~708 to ~2,468 Torr. From these measurements, we estimated VO2 to be 7.9 × 10-5 and 7.3 × 10-6 ml O2 · cm-3 · min-1, respectively. Although the absolute values varied, both methods of determining VO2 showed that VO2 was consistently reduced under the more extreme hyperoxic conditions compared with control PO2 values.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

Neuronal tissue PO2 has been measured in the intact CNS during HBO2 (24, 27, 51, 61), and in brain slices at normobaric pressure (3-5, 19-21, 28, 50, 56); however, this is the first study to systematically study PO2 gradients in brain slices during HBO2. We found that, under conventional brain slice control conditions (95% O2), PO2 measured in the solitary complex decreased with increasing recording depth to a minimum PO2 value at the core of the slice that was still ~10-fold higher than normal cerebral PO2 in vivo, which has been reported to range from 10 to 34 Torr (Table 1). In fact, at an aCSF PO2 of 708 Torr, PO2 at the core of the slice approximated PO2 measured in the CNS of rats breathing 100% oxygen at PB of >2 ATA (27). Furthermore, tissue PO2 increased linearly with aCSF PO2 from 156 to 2,468 Torr, to levels that are known to result in CNS O2 toxicity in whole animals (1, 25, 58). This range of HBO2 has been reported to depolarize solitary complex neurons in brain stem slices after <= 10 min of exposure (12, 45, 46). At PO2 values >1,200 Torr, the difference in PO2 from the surface to tissue core (i.e., Delta PO2) decreased to the extent that Delta PO2 measured in metabolically active tissue exposed to hyperoxic medium no longer differed from Delta PO2 made in metabolically poisoned tissue. This difference was attributed to a reduction in VO2, suggesting that the higher levels of HBO2 may decrease cellular respiration in brain slices, as previously reported (1, 11).

Critique of Methods

Initially, we measured oxygen with platinum needle electrodes (12); however, these electrodes showed poor resolution between 50-µm steps in tissue (not shown). This resolution problem likely resulted from the high rate of VO2 by the electrode, as signified by the large current generated per Torr oxygen (7.87 nA/Torr), which then depleted oxygen from the area around the electrode tip, thus blunting the PO2 difference per 50 µm. Therefore, we switched to carbon fiber electrodes of the type typically used for the voltametric detection of neurotransmitters and metabolites around synapses (41). Polarographic electrodes can measure oxygen at a polarization potential of -0.6 V with minimal interference from oxidizable substances (e.g., neurotransmitters are typically oxidized at potentials of 0.2-0.8 V), and, because these electrodes are small and of high resistance, they are ideal for measuring oxygen in brain slices (28). Only carbon fiber electrodes were used in this study; as illustrated in Fig. 2B, these electrodes produced a current that was linearly proportional to oxygen concentration at both normobaric and hyperbaric pressure.

As was the case in previous studies (4, 19, 21), there was considerable variability in our PO2 measurements. This variability likely resulted from error in the estimated tissue depth due to tissue dimpling as the electrode penetrated the slice, uneven brain slice thickness, or tissue debris on the electrode tip that reduced the tip surface available for the reduction of oxygen (10). Likewise, tissue debris on the electrode tip likely accounted for offsets that occurred in about one-half of the electrodes used. Slow tissue potential changes or direct current shifts, which can influence PO2 measurements,3 were presumed to be small in the solitary complex during exposure to the same hyperoxic conditions, compared with the -0.6-V polarizing potential (12, 45, 46). Furthermore, it has also been shown that, when using a low-resistance remote reference, the effects of any slow tissue potential changes on the polarizing voltage were negligible (37). More stable methods of measuring PO2, such as with a Clark-style oxygen electrode (17) or the optical phosphorescence method (32), were not used because of the constraints of doing such measurements inside a hyperbaric chamber at PB >1 ATA.

PO2 Profiles in aCSF

At the gas-liquid interface, oxygenated aCSF was in contact with an oxygen-free helium atmosphere. Oxygen, according to Le Châtelier's principle, will diffuse from the aCSF down its chemical gradient into the overlying chamber atmosphere, leaving behind a graded layer of PO2 in the aCSF. As expected, PO2 was minimum at the aCSF surface and increased with increasing depth of aCSF. However, the depth at which a measurable diffusional loss of oxygen to the chamber atmosphere no longer occurred was consistently ~450 µm regardless of the media PO2. In addition, our results indicate that submerged brain slices are oxygenated by the diffusion of oxygen from aCSF of approximately ±200 µm to the tissue surface. We observed that, at medium PO2 values >= 708 Torr, oxygen diffusion into the brain slice resulted in a 35-40% drop in PO2 from bulk aCSF to slice surface. Previous studies have noted similar diffusion layers, sometimes referred to as unstirred layers in brain slices (4, 19, 21, 39) and in the brain stem spinal cord preparation (52).

If we assume that the oxygen diffusion gradient in the bath is identical with or without the tissue slice present, then these results suggest that oxygenation of the top surface of the slice is limited by the following two factors: 1) the diffusion of oxygen into the helium atmosphere and 2) the depth of the aCSF overlying the slice. Thus maintaining <450 µm of perfusate over the slice could potentially limit oxygenation at the upper surface of the slice. In our preparation, however, the brain slice was always positioned >450 µm deep to the bath surface.

PO2 Profiles in Brain Slices

Measurements of PO2 through 300-µm-thick brain slices exposed to medium PO2 ranging from 156 to 2,468 Torr showed that, although oxygen tension decreased with increasing recording depth in tissue, PO2 measured at the tissue surface (0 µm) and tissue core (150 µm) increased linearly as medium PO2 increased. As expected, these results indicate that the oxygen diffusion coefficient in tissue did not change with medium PO2 or diffusion distance. Furthermore, the magnitude of the oxygen gradient in aCSF from <= 200 µm above or below the slice was roughly equivalent to the oxygen gradient in the outer 100-µm layers of tissue. A similar observation was previously reported in the neonatal rat brain stem spinal cord preparation (52).

The majority of our tissue PO2 profiles were symmetrical, with the minimum PO2 value measured approximately at the center of the slice. Some studies conducted at normobaric pressure came to similar conclusions (19-21). In contrast, investigators who used the interface slice preparation, with an overlying atmosphere of 95% O2, reported that diffusion from the upper surface dominated and resulted in a minimum PO2 near the bottom of the slice (28).

Control PO2 at Normobaric Pressure

When slices were submerged in aCSF equilibrated with 95% O2, we measured a minimum PO2 of 291 ± 83 Torr at the center of the slice. A similar minimum PO2 value of 187.2 ± 11 Torr (n = 2) was measured at a depth of 150 µm in 320-µm-thick guinea pig cortical slices submerged in aCSF equilibrated with 95% O2 at 1 ATA (19). Likewise, although variability between brain slice preparations and experimental parameters makes direct comparison of absolute slice PO2 difficult, minimum control PO2 values reported here were similar to values measured in 400- to 450-µm thick brain slices positioned in the interface preparation; these values ranged from ~150 to 280 Torr (28, 50).

Minimum PO2 values measured in our slice preparation and in others (19, 28, 50) when incubated with conventional control solution (95% O2, PB of 1 ATA) were ~10-fold greater than PO2 values measured in vivo (9, 23, 24, 27, 51, 61). This indicates that most brain slice studies are performed under hyperoxic conditions at normobaric pressure, thus raising the concern that neuronal activity may be affected by an increased production of ROS. It has been shown that the degree of tissue damage resulting from lipid peroxidation was significantly increased in brain slices incubated in 95% O2 compared with 21% O2 at normobaric pressure (33, 54). Bingmann and colleagues (3, 5) found neurons in hippocampal slices incubated in ~21% O2 depolarized and increased their firing rate when exposed to 100% O2, indicating that the high PO2 of brain slice control medium (i.e., normobaric hyperoxia) can, in fact, alter cellular activity. The activity of hippocampal neurons in 21% O2 was not considered a response to hypoxia (in the brain slice preparation, hypoxia is typically mimicked by Fo2 values of 10-15% O2 at PB of 1 ATA), however, because the activity of hippocampal neurons exposed to hypoxia was quite different (15, 36). Normobaric hyperoxia has also been shown to alter neuronal function in hypothalamic slices (55) and in the carotid body (43), and these responses were attributed to increased ROS.

Characterization of an optimal medium PO2 has proven to be important for thin tissue preparations like neuronal cell cultures. For example, in cultures of neocortical and hippocampal neurons, the optimal medium PO2, based on cell growth and viability, was determined to be 9% O2 at PB of 1 ATA (~68 Torr) (7, 30). Neuritogenesis of cultured hippocampal neurons was also improved by the addition of antioxidants (vitamin E, vitamin A, and linolenate) to the incubation medium (7). These results suggest that the optimal medium PO2 for an in vitro tissue preparation must balance tissue oxygen requirements with the otherwise toxic oxidative effects of excess oxygen.

Clearly, medium PO2 affects both neuronal viability and excitability. For this reason, it is important that in vitro experimental conditions match, as closely as possible, in vivo conditions (i.e., optimum Fo2 of the perfusion medium should produce a PO2 at the core of the brain slice that ranges between 10 and 34 Torr). In our study, when medium PO2 was reduced from control to 21% O2, although slice surface PO2 was still hyperoxic, the minimum PO2 values, which averaged 40 ± 17 Torr, more closely resembled PO2 values measured in the CNS and CSF of whole animals (Table 1). Alternatively, antioxidants can be added to the medium to provide protection from ROS when using 95% O2 (6, 7, 34). Subsequent electrophysiological studies of solitary complex neurons in 300-µm-thick brain slices are required to confirm, however, that cells remain viable and healthy in this preparation at this lower level of control PO2.

Metabolically Poisoned Tissue

VO2 is another important factor that must be considered when determining the optimum brain slice control PO2. Bingmann et al. (4) reported that cellular VO2 increased when the incubation medium PO2 of 300-µm-thick hippocampal slices increased from 150 to 600 Torr. The authors suggested that, at normobaric pressure and a medium PO2 of 150 Torr, cell respiration was limited by oxygen availability such that an increase in medium PO2 resulted in an increase in VO2. However, the extent to which brain slice VO2 is directly dependent on medium PO2 is not known under conditions of HBO2. For these reasons, we measured PO2 in brain slices equilibrated with aCSF having PO2 values that ranged from 156 to 2,468 Torr; then, for comparison, PO2 measurements were repeated in metabolically poisoned tissue equilibrated with the same range of PO2 values. Oxygen profiles in metabolically active slices showed that, as PO2 increased from 708 to 1,657 Torr, the Delta PO2 approached a minimum, at 1,657 Torr, that was significantly different from the Delta PO2 at 708 Torr and not different from Delta PO2 values in metabolically poisoned tissue. At PO2 greater than 1,657 Torr, Delta PO2 values of oxygen profiles remained similar in magnitude to Delta PO2 values made in metabolically poisoned tissue. In addition, calculated VO2 under control conditions (95% O2) was 1.8 ± 0.2 × 10-3 ml O2 · cm-3 · min-1. A comparable VO2 of 3.38 ± 0.31 × 10-2 ml O2 · cm-3 · min-1 was measured in 500-µm-thick slices of guinea pig olfactory cortex equilibrated with 95% O2 (20). During HBO2-3, VO2 was reduced to 9.3 ± 1.3 × 10-4 ml O2 · cm-3 · min-1. Furthermore, although absolute VO2 values varied, this trend was maintained when VO2 was assumed to be dependent on medium PO2 (see APPENDIX). Together, these results suggest that the higher levels of HBO2 reduced metabolism in 300-µm-thick slices.

The mechanism by which HBO2 may reduce brain slice metabolism is not clear but likely involves the increased production of ROS and the oxidation of mitochondrial enzymes and/or cofactors, including alpha -lipoic acid, cytochrome c, flavin nucleotides, and ubiquinone (1, 11). Furthermore, neuronal responses to HBO2 depend on the duration of the HBO2 exposure. Previous electrophysiological recordings show that short (<= 10 min) bouts of HBO2 increase neuronal activity (12, 45, 46); it is well known that VO2 increases in conjunction with neuronal activity (42); however, in this study, we presented evidence suggesting that 30 min or more of exposure to the same HBO2 actually reduced VO2. Future studies focusing on the details regarding the dose dependence of HBO2 sensitivity may be necessary.

In conclusion, oxygen tension in the submerged brain slice during normobaric hyperoxia and HBO2 was a complex function that was dependent on several experimental conditions, including ambient PO2, depth of slice in the tissue bath, and VO2. Our findings show that PO2 in the solitary complex of the 300-µm-thick brain slice submerged in control medium (95% O2 at PB of ~1 ATA) was hyperoxic compared with the in vivo CNS. When exposed to HBO2, tissue PO2 increased to oxygen tensions that corresponded with cerebral PO2 values measured in vivo under conditions that result in symptoms of CNS O2 toxicity (1, 25, 58). Our results also suggest that metabolism decreased during high levels of HBO2, which was consistent with previous observations (1, 11) and suggests that there may be a metabolic component to the mechanism of HBO2-induced neuronal sensitivity.


    APPENDIX
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

By assuming one-dimensional diffusion and uniform boundary conditions across the surface of the slice, D can be approximated by the solution to Fick's second law of diffusion (8)
P<SC>o</SC><SUB><IT>2</IT></SUB><IT>≈</IT>P<SUB><IT>1</IT></SUB><IT>+</IT><FR><NU><IT>4</IT>(P<SUB><IT>0</IT></SUB><IT>−</IT>P<SUB><IT>1</IT></SUB>)</NU><DE><IT>&pgr;</IT></DE></FR> sin <FENCE><FR><NU><IT>&pgr;X</IT></NU><DE><IT>L</IT></DE></FR></FENCE> exp<FENCE>−<IT>D</IT><FENCE><FR><NU><IT>&pgr;</IT></NU><DE><IT>L</IT></DE></FR></FENCE><SUP><IT>2</IT></SUP><IT>t</IT></FENCE>
where P0 is the measured PO2 at a depth of 150 µm in tissue equilibrated with a medium PO2 of 708 Torr, P1 is the measured PO2 at a depth of 150 µm in tissue equilibrated with a medium PO2 of 156 Torr, PO2 is calculated to be the mean of P0 and P1, X is the recording depth (150 µm), L is tissue thickness (300 µm), and t is time, in seconds, to reach PO2. We calculated D at a PO2 halfway between two steady-state conditions because, presumably, O2 flux would be maximum. We estimated D to be 1.3 × 10-6 cm2/s. Our estimated D was smaller than the D calculated from PO2 measurements in 1,000-µm-thick slices of cat cortex equilibrated with a similar initial PO2, 1.54 × 10-5cm2/s (21).

Our results suggest that slice VO2 was dependent on PO2 of the bathing medium. By assuming that VO2 was dependent on PO2, we approximated VO2 from PO2 measurements made at a depth of 150 µm in a brain slice equilibrated with regular aCSF while switching medium PO2 from 708 to 156 or 2,468 Torr. With the same boundary conditions as before, VO2 was approximated graphically from the equation (8)
&phgr;(&bgr;)≈−P<SC>o</SC><SUB><IT>2</IT></SUB><IT>+</IT><FENCE><FR><NU><IT>2</IT>P<SUB><IT>1</IT></SUB></NU><DE>sinh (<IT>&bgr;L</IT>)</DE></FR></FENCE> sinh <FENCE><FR><NU><IT>&bgr;L</IT></NU><DE><IT>2</IT></DE></FR></FENCE>

<IT>+</IT><FENCE><FR><NU><IT>4</IT>P<SUB><IT>0</IT></SUB></NU><DE><IT>&pgr;</IT></DE></FR><IT>−</IT><FR><NU><IT>4&pgr;</IT>P<SUB><IT>1</IT></SUB></NU><DE><IT>&pgr;<SUP>2</SUP>+</IT>(<IT>&bgr;L</IT>)<SUP><IT>2</IT></SUP></DE></FR> cosh <FENCE><FR><NU><IT>&bgr;L</IT></NU><DE><IT>2</IT></DE></FR></FENCE></FENCE> exp<FENCE>−<IT>D</IT><FENCE><FR><NU><IT>&pgr;</IT></NU><DE><IT>L</IT></DE></FR></FENCE><SUP><IT>2</IT></SUP><IT>t</IT></FENCE>
where phi (beta ) = beta  = <RAD><RCD><A><AC>V</AC><AC>˙</AC></A>/<IT>D</IT></RCD></RAD>, P0 is the measured PO2 at a depth of 150 µm into a slice equilibrated with a medium PO2 of 708 Torr, P1 is the measured PO2 at a depth of 150 µm into a slice equilibrated with either 156 or 2,468 Torr, PO2 is equal to the mean of P0 and P1, t is time, in seconds, to reach PO2, and D is 1.3 × 10-6 cm2/s. To convert VO2 to units of milliliters O2 per cubic centimeters per minute, the approximated VO2 was multiplied by the oxygen solubility coefficient of cat brain, 0.0144 ml O2 · cm3 tissue-1 · atm-1 (21). Switching medium PO2 from 708 to 156 or from 708 to 2,468 Torr resulted in estimated VO2 of 7.9 × 10-5 or 7.3 × 10-6 ml O2 · cm3 tissue-1 · min-1, respectively.


    ACKNOWLEDGEMENTS

We acknowledge the assistance of Drs. L. Turyn and T. Svobodny for their help in calculating D and VO2, Michael R. Rayle for statistical assistance, Dr. C. Jiang (Georgia State University) for assistance with construction and use of the carbon fiber electrodes, and Phyllis Douglas for technical assistance.


    FOOTNOTES

The research was supported, in part, by National Heart, Lung, and Blood Institute Grant R01-HL-56683, Wright State University (WSU) Alpha Grant Program (Kettering Foundation), and the Office of Naval Research Grant N000140110179. D. K. Mulkey was supported by the WSU Biomedical Sciences PhD program.

Address for reprint requests and other correspondence: J. B. Dean, Dept. of Physiology and Biophysics, Rm. 160 Biological Sciences Bldg., 3640 Colonel Glenn Hwy, Wright State Univ., Dayton, OH 45435 (E-mail: jay.dean{at}wright.edu).

1 At sea level, PB is 1 ATA or 760 Torr. Other commonly used pressure equivalents include 0.099 MPa (SI unit), 14.7 pounds per inch2, and 1.01 bar.

2 In Dayton, OH, the PB averaged 745 ± 2 (SE) Torr. When determining medium PO2, the vapor pressure of water, which is ~48 Torr at 37 ± 0.3°C (60), was not subtracted from PB.

3 Lehmenkuhler et al. (37) reported that, when making PO2 measurements in excitable tissue, slow tissue potential changes or direct current shifts resulting from oxygen-induced excitation may mimic changes in PO2 by interfering with the polarizing voltage at the oxygen electrode.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1 November 2000; accepted in final form 29 December 2000.


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J APPL PHYSIOL 90(5):1887-1899
8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society


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