Brain oxygenation and CNS oxygen toxicity after infusion of ...

J Appl Physiol 113: 224 –231, 2012. First published May 3, 2012; doi:10.1152/japplphysiol.00308.2012.

Brain oxygenation and CNS oxygen toxicity after infusion of perfluorocarbon emulsion Ivan T. Demchenko,1,2 Richard T. Mahon,4 Barry W. Allen,1,2 and Claude A. Piantadosi1,2,3 1

Center for Hyperbaric Medicine and Environmental Physiology, Duke University Medical Center, Durham, North Carolina; Department of Anesthesiology, Duke University Medical Center, Durham, North Carolina; 3Department of Medicine, Duke University Medical Center, Durham, North Carolina; and 4Undersea Medicine Department, Naval Medical Research Center, Silver Spring, Maryland

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Submitted 9 March 2012; accepted in final form 1 May 2012

Demchenko IT, Mahon RT, Allen BW, Piantadosi CA. Brain oxygenation and CNS oxygen toxicity after infusion of perfluorocarbon emulsion. J Appl Physiol 113: 224 –231, 2012. First published May 3, 2012; doi:10.1152/japplphysiol.00308.2012.—Intravenous perfluorocarbon (PFC) emulsions, administered with supplemental inspired O2, are being evaluated for their ability to eliminate N2 from blood and tissue prior to submarine escape, but these agents can increase the incidence of central nervous system (CNS) O2 toxicity, perhaps by enhancing O2 delivery to the brain. To assess this, we infused a PFC emulsion (Oxycyte, 6 ml/kg iv) into anesthetized rats and measured cerebral PO2 and regional cerebral blood flow (rCBF) in cortex, hippocampus, hypothalamus, and striatum with 100% O2 at 1, 3, or 5 atmospheres absolute (ATA). At 1 ATA, brain PO2 stabilized at ⬎20 mmHg higher in animals infused with PFC emulsion than in control animals infused with saline, and rCBF fell by ⬃10%. At 3 ATA, PFC emulsion raised brain PO2 ⬎70 mmHg above control levels, and rCBF decreased by as much as 25%. At 5 ATA, brain PO2 was ⱖ159 mmHg above levels in control animals for the first 40 min but then rose sharply; rCBF showed a similar profile, reflecting vasoconstriction followed by hyperemia. Conscious rats were also pretreated with PFC emulsion at 3 or 6 ml/kg iv and exposed to 100% O2 at 5 ATA. At the lower dose, 80% of the animals experienced seizures by 33 min compared with 50% of the control animals. At the higher dose, seizures occurred in all rats within 25 min. At these doses, administration of PFC emulsion poses a clear risk of CNS O2 toxicity in conscious rats exposed to hyperbaric O2 at 5 ATA. brain oxygen partial pressure; cerebral blood flow; central nervous system oxygen toxicity TWO APPROACHES are favored for prevention of decompression sickness (DCS) or arterial gas embolism due to rapid decompression or for use as adjuncts to primary therapies for treatment or mitigation of these conditions. One method, breathing pure O2 prior to decompression (O2 prebreathing), was initially developed to reduce the risk of DCS in military aviation (12, 13) and has been adopted for use by underwater divers (15), as well as for preparation of astronauts for extravehicular excursions in the hypobaric microenvironment of a spacesuit (22). The benefit of O2 breathing prior to decompression has been demonstrated in humans (15) and animals (1, 25) and is attributed to the accelerated elimination of inert gas from blood and tissue. The second approach is to administer a perfluorocarbon (PFC) emulsion intravenously before or after decompression to increase the capacity of the blood plasma to carry gases in

Address for reprint requests and other correspondence: B. W. Allen, Center for Hyperbaric Medicine and Environmental Physiology, Duke Univ. Medical Center, Durham, NC 27710 (e-mail: [email protected]). 224

solution and transport them to the lungs for elimination. Originally developed as artificial blood substitutes, the solubilities and diffusivities of gases in a PFC emulsion are many times greater than in water or physiological salt solutions (11); these properties accelerate the rate at which O2 is transferred to tissues and inert gases are removed (24). Most PFC emulsions have average diameters of 0.2 ␮m compared with 5–7 ␮m for erythrocytes, and particles of this size can access spaces between red blood cells and vessel walls and perfuse even constricted microvessels (30). O2 breathing can be used alone to prevent or mitigate injury due to decompression (20), but when a PFC emulsion is used for this purpose, it is administered in combination with O2, since its effectiveness in eliminating inert gases depends on the diffusion gradient between the tissues and the alveoli. In early studies, mortality decreased when supplemental inspired O2 was used with first-generation PFC emulsions (Fluosol DA and Fluosol-43, Green Cross, Osaka, Japan) in rats and hamsters after rapid decompression (17, 18, 27). Recent studies in swine (3) evaluated O2 administration combined with a second-generation PFC emulsion (Oxygent, Alliance Pharmaceutical, San Diego, CA), a mixture of perfluorooctyl bromide and perfluorodecyl bromide emulsified with egg yolk phospholipid, administered intravenously before (21) or after (9) rapid decompression. The greatest benefit occurred when this drug was given after decompression (4). Oxygent with inhaled O2 has been shown to reduce numbers of bubbles appearing after decompression (28) or hasten their disappearance (23). A third-generation PFC emulsion (Oxycyte, tert-butyl perfluorocyclohexane emulsified with egg yolk phospholipid), developed by Leland Clark, is undergoing clinical trials (Oxygen Biotheraputics, Research Triangle Park, NC). The O2carrying capacity of pure Oxycyte (without the emulsifying agent) is 43 ml O2/100 ml at 37°C and 760 mmHg O2 (30), and it has been studied under normobaric (30) and hyperbaric conditions (19). A comprehensive review of the physical and chemical properties of this agent is available elsewhere (26). Although PFC emulsions have been reported to dramatically reduce mortality and delay the onset of DCS, high rates of central nervous system (CNS) toxicity have also been reported (21). The solubility of O2 is ⬃20 times greater per unit volume in Oxygent than in blood plasma; therefore, in hyperbaric O2 (HBO2), potentially toxic concentrations of O2 could be dissolved in this agent and delivered to vulnerable brain regions, despite protective cerebral vasoconstriction. In addition, we have shown that the vasoconstrictor response to hyperoxia is reversed by HBO2-stimulated nitric oxide production, and the http://www.jappl.org

Brain PO2, CBF, And O2 Toxicity in HBO2 With PFC

resulting increase in perfusion accelerates O2 delivery to the brain (6). This secondary increase in cerebral blood flow (CBF), as well as the HBO2-related EEG spikes that follow, are abolished by nitric oxide synthase inhibition but reappear after treatment with L-arginine (5). A high incidence of CNS toxicity in animals treated with Oxygent and exposed to HBO2 at 5 ATA (21) demonstrates the potential risk of using such agents under these conditions. Quantitative data are needed to guide the development of safe protocols for use of this and other PFC emulsions in HBO2. The goal of the present study was to measure the effects of intravenous Oxycyte on temporal profiles of PO2 and blood flow in four brain regions critical to seizure development to test the hypothesis that the PFC emulsion increases brain PO2 in HBO2. We also sought to define approximate limits for dose, time, and pressure for use of a biocompatible PFC emulsion in HBO2 without evoking CNS O2 toxicity. METHODS

Animals. Anesthetized and awake male Sprague-Dawley rats (Charles River; 341 ⫾ 27 g body wt) were used in a protocol approved by the Duke University Institutional Animal Care and Use Committee. Anesthesia was induced with urethane (750 mg/kg ip) and ␣-chloralose (75 mg/kg ip). As described elsewhere (7), both femoral arteries and one femoral vein were catheterized for monitoring blood pressure, taking samples, and administering drugs. The trachea was intubated, and the animals were initially ventilated with air in ambient laboratory conditions (“room air”). Platinum needle electrodes, for measurement of PO2 or CBF, were inserted stereotactically through burr holes located over the striatum, hippocampus, hypothalamus, and parietal cortex; electrode placement was confirmed postmortem. Only rats that were deeply anesthetized, verified by observation of the cardiovascular responses to toe pinch, were given pancuronium bromide (0.5 mg/kg iv). Anesthesia and immobilization were maintained by intravenous administration of one-quarter of the initial doses of the anesthetic and paralytic each hour or as necessary. Previous control studies indicate that this regimen of supplemental anesthesia is adequate for these HBO2 exposures. Physiological measurements. Arterial blood pressure was measured continuously and integrated to obtain mean arterial blood pressure. Arterial PO2 (PaO2), PCO2 (PaCO2), and pH were determined in blood samples (IL 1306 blood gas/pH analyzer) in rats breathing air; ventilation rate was adjusted to keep blood gases in the physiological

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range before compression and kept constant thereafter. Rectal temperature was monitored continuously and held at 37 ⫾ 0.5°C by a thermostatically controlled heating pad. Brain PO2 was measured by the needle electrodes (7), which were calibrated before and after each experiment in three artificial cerebrospinal fluid buffers at 1 ATA and 37°C, equilibrated with 100% N2 (0% O2), air (21% O2), and 100% O2; the PO2 of each buffer was verified with the blood gas analyzer. If initial and final calibrations diverged by ⬎10%, data from that electrode were excluded. Correlation between electrode output and cerebral PO2 in HBO2 was determined from the calibration curve by linear regression. Regional CBF (rCBF) was measured in HBO2 by H2 clearance (7), for which the PO2 electrodes were poised at an oxidizing potential as 2.5% H2 in air was introduced through the respirator for 60 s. H2-washout curves were captured using WINDAQ software (D-1200 AC, DATAQ Instruments). Absolute rCBF (ml·g⫺1·min⫺1) was calculated by the initial slope method using Mathematica 3.0 software (Wolfram Research) and expressed as percentage of baseline levels in animals breathing room air prior to infusion of PFC emulsion. HBO2 exposure. Anesthetized rats, along with the stereotaxic frame, respirator, blood pressure transducer, heating pad, and infusion pump, were placed in a hyperbaric chamber (Duke Center for Hyperbaric Medicine and Environmental Physiology). After a 60-min stabilization period at 1 ATA, during which blood gas values remained normal, PO2 or rCBF was measured in the four brain regions simultaneously. The respirator was supplied with 100% O2, and chamber air pressure was left at 1 ATA or raised to 3 or 5 ATA at 0.6 ATA/min. The duration of hyperoxic exposures for anesthetized rats was 60 min, and cerebral PO2 was measured continuously or CBF was measured every 10 min. Experimental groups. Seventeen groups of experiments were conducted: anesthetized rats were used in 14 experiments and conscious rats were used in 3 experiments (Table 1). In experiments 1– 8, brain PO2 was measured in anesthetized animals after intravenous administration of 0.9% NaCl (saline controls) or PFC emulsion (Oxycyte, 6 ml/kg) during ventilation with room air or 100% O2 at 1, 3, or 5 ATA. In experiments 9 –14, rCBF was measured in anesthetized animals during ventilation with 100% O2 at 1, 3, or 5 ATA after infusion of saline (controls) or PFC emulsion (6 ml/kg). In experiments 15–17, unrestrained, awake rats were given saline (6 ml/kg) or PFC emulsion (3 or 6 ml/kg) before exposure to HBO2 at 5 ATA for 45 min, and latency to clonic-tonic seizures was determined by direct observation. PFC emulsion or saline was infused immediately before HBO2 exposure in the catheterized femoral vein in anesthetized rats or in a tail vein in briefly restrained awake animals.

Table 1. Hyperoxic exposures and measured parameters for anesthetized and awake rats Group No.

n

Room Air

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

6 7 7 7 8 8 9 9 7 7 8 8 9 9 10 10 10

X X

100% O2

1 1 3 3 5 5 1 1 3 3 5 5 5 5 5

ATA, ATA, ATA, ATA, ATA, ATA, ATA, ATA, ATA, ATA, ATA, ATA, ATA, ATA, ATA,

60 60 60 60 60 60 60 60 60 60 60 60 45 45 45

min min min min min min min min min min min min min min min

Brain PO2

rCBF

Seizure Latency

X X X X X X X X

Saline, ml/kg

PFC Emulsion, ml/kg

6 6 6 6 6 6 6 6 X X X X X X

6 6 6 6 6 6 X X X

rCBF, regional cerebral blood flow; PFC, perfluorocarbon; ATA, atmosphere absolute. J Appl Physiol • doi:10.1152/japplphysiol.00308.2012 • www.jappl.org

6 3 6

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Fig. 1. Brain PO2 in 100% O2 at 1 ATA. In all experiments, perfluorocarbon (PFC) emulsion or saline (6 ml/kg) was infused intravenously immediately before respiratory gas was switched from room air. PO2 was significantly higher in animals treated with PFC emulsion at almost all time points and trended slightly downward over 60-min hyperoxic exposure compared with lower and more constant values in saline-infused control animals. *P ⬍ 0.05 vs. saline at the same time point.

Data analysis. Values from groups of animals are expressed as means ⫾ SD. Absolute and percent changes in PO2 or CBF during hyperoxic exposure were compared with baseline values obtained in room air prior to treatment by one-way ANOVA followed, if needed, by Fisher’s protected least significant difference test. For comparisons between saline- and PFC emulsion-treated groups at the same time points, paired t-tests were used. P ⬍ 0.05 is accepted as significant.

RESULTS

Effects of intravenous PFC emulsion on brain PO2 and CBF in rats breathing room air. Baseline PO2 before PFC emulsion administration averaged 26 ⫾ 8.7 mmHg in the hypothalamus, 24 ⫾ 7.4 mmHg in the striatum, 21 ⫾ 7.3 mmHg in the hippocampus, and 23 ⫾ 8.2 mmHg in the cortex. After intravenous infusion of PFC emulsion (6 ml/kg), neither

Fig. 2. Cerebral blood flow (CBF) in 100% O2 at 1 ATA. Regional CBF decreased after infusion of PFC emulsion (shaded bars) but was not seen in control animals (solid bars). ⫹P ⬍ 0.05 vs. animals breathing room air before intravenous saline infusion. *P ⬍ 0.05 vs. saline at the same time point.

J Appl Physiol • doi:10.1152/japplphysiol.00308.2012 • www.jappl.org


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Fig. 3. Brain PO2 in hyperbaric O2 (HBO2) at 3 ATA. PFC emulsion or saline (6 ml/kg) was administered intravenously immediately before beginning of compression. At this level of hyperoxia, downward trend seen with PFC emulsion at 1 ATA was amplified. *P ⬍ 0.05 vs. saline.

brain PO2 nor rCBF differed significantly from baseline values. Brain PO2 and CBF in 100% O2 at 1 ATA. After the respiratory gas was switched to 100% O2 but before infusion of saline or PFC emulsion, brain PO2 rose and remained significantly above baseline in each brain region, peaking between 83 and 108 mmHg, and these levels were unchanged by subsequent saline infusion. However, when administration of PFC emulsion was combined with 100% O2, brain PO2 ranged from 103 to 133 mmHg (Fig. 1). O2, with or without saline infusion,

reduced rCBF by ⬍10% compared with baseline values in air, whereas O2 together with PFC emulsion decreased rCBF by 13–19% after 30 min, and this enhanced cerebral vasoconstriction persisted for the rest of the 60-min exposure (Fig. 2). Brain PO2 and rCBF in HBO2 at 3 ATA. Temporal profiles of brain PO2 responses to HBO2 at 3 ATA with and without PFC emulsion were biphasic, increasing during compression and then trending gradually downward throughout the 60-min exposure. In animals pretreated with PFC emulsion (6 ml/kg), brain PO2 rose minimally in the hippocampus and maximally in

Fig. 4. CBF in HBO2 at 3 ATA. CBF decreased progressively, more in PFC emulsion-treated (shaded bars) than control (solid bars) animals. #P ⬍ 0.05 vs. animals breathing room air before intravenous saline. ⫹P ⬍ 0.05 vs. animals breathing room air before intravenous PFC emulsion. *P ⬍ 0.05 vs. saline.


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Fig. 5. Cerebral PO2 in HBO2 at 5 ATA. Attenuated decreases in brain PO2 were observed in all 4 brain regions in the first half of exposure to HBO2 at 5 ATA, interrupted after 30 min by steep increases, most notably in PFC emulsion-treated animals. *P ⬍ 0.05 vs. saline.

the hypothalamus, augmenting brain oxygenation at the beginning of hyperbaric exposure by ⬃23% above that resulting from HBO2 alone (Fig. 3). After 60 min in HBO2, rCBF in the control rats decreased 16 –24% below preexposure levels, whereas infusion of PFC emulsion lowered rCBF by 23–32%, with the greatest changes in the striatum and hippocampus (Fig. 4). Protective vasoconstriction was maintained throughout these experiments. Brain PO2, rCBF, and seizures in HBO2 at 5 ATA. In anesthetized animals with and without intravenous PFC emul-

sion, brain PO2 displayed a triphasic pattern, increasing during compression, decreasing slightly as 5 ATA was reached, and then increasing further after 30 min (Fig. 5). This pattern was amplified in rats that received PFC emulsion. Initially, rCBF decreased but returned to control levels or above, usually within 30 – 40 min of HBO2 exposure, coinciding with escape from protective vasoconstriction due to a decrease in cerebrovascular tone (Fig. 6). In separate experiments with conscious rats exposed to O2 at 5 ATA for 45 min, seizure incidence increased (Fig. 7A) and

Fig. 6. CBF in HBO2 at 5 ATA. Escape from protective vasoconstriction was observed in all animals after 30 min; PFC emulsion (shaded bars) intensified this response compared with saline (solid bars). #P ⬍ 0.05 vs. animals breathing room air before intravenous saline. ⫹P ⬍ 0.05 vs. animals breathing room air before PFC emulsion. *P ⬍ 0.05 vs. saline.



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Fig. 7. Seizure latency in awake rats. PFC emulsion increased incidence of seizures (A) and shortened seizure latency (B) in a dose-dependent manner. *P ⬍ 0.05 vs. saline.

time to clonic-tonic seizures decreased significantly after pretreatment with PFC emulsion (6 ml/kg) compared with control animals (Fig. 7B). Rats given PFC emulsion at the lower dose (3 ml/kg) also demonstrated shortened seizure latency (Fig. 7B), but differences from control values were not significant. DISCUSSION

This is the first study to quantify changes in tissue PO2 and blood flow concurrently in multiple brain regions at different levels of hyperoxia after intravenous administration of a thirdgeneration PFC emulsion. In rats breathing room air, administration of PFC emulsion did not significantly alter brain PO2 or rCBF in the regions studied. During ventilation with 100% O2 at 1, 3, and 5 ATA, the PFC emulsion enhanced brain PO2 Table 2. Calculated alveolar and blood gases and blood O2 content in HBO2 1 ATA

3 ATA

5 ATA

Alveolar gas equation: PAO2 ⫽ [FIO2 ⫻ (Patm ⫺ PH2O)] ⫺ {PaCO2[1 ⫺ FIO2 (1 ⫺ RQ)]/RQ} FIO2, % Patm, mmHg PH2O, mmHg PaCO2, mmHg RQ PAO2, mmHg PaO2, mmHg

100 760 47 40 0.8 673 606

100 2,280 47 40 0.8 2,193 1,974

100 3,800 47 40 0.8 3,713 3,342

O2 content equation: CaO2 ⫽ [Hb] ⫻ (1.34 ml O2/g Hb) ⫻ SaO2 ⫹ PaO2 ⫻ (0.003 ml O2 䡠 mmHg⫺1 䡠 dl⫺1) [Hb], g/dl O2 binding by Hb, ml/g SaO2, % PaO2, mmHg O2 solubility in plasma, ml O2 䡠 mmHg⫺1 䡠 dl⫺1 CaO2, ml/dl

16 1.34 1 606

16 1.34 1 1,974

16 1.34 1 3,342

0.003 23.4

0.003 27.9

0.003 32.4

HBO2, hyperbaric O2; PAO2, alveolar PO2; FIO2, inspiratory O2 fraction; Patm, atmospheric pressure; PH2O, water vapor pressure; PaCO2, arterial PCO2; RQ, respiratory quotient; PaO2, arterial PO2; CaO2, arterial O2 content; [Hb], hemoglobin concentration; SaO2, arterial O2 saturation. Values in boldface are results carried over into the next calculation.

and altered CBF compared with rats breathing room air before infusion of PFC emulsion and saline control rats exposed to the same levels of HBO2. In rats breathing 100% O2 at 1 and 3 ATA for 60 min, rCBF was lowered to a greater extent by the PFC emulsion than by hyperoxia, with no release of cerebral vasoconstriction. However, in animals breathing O2 at 5 ATA, the circulating PFC emulsion significantly increased brain PO2 and shortened the period of protective cerebral vasoconstriction. Therefore, in animals breathing O2 at 1 and 3 ATA, the PFC emulsion appears to increase brain PO2 through increases in dissolved blood O2 content. At 5 ATA, however, increases in brain PO2 after infusion of PFC emulsion appear to be the result of a combination of elevated arterial O2 content (CaO2) and augmented rCBF due to escape from protective vasoconstriction. The relative effects of these two processes are demonstrated in step-wise fashion in the separate groups of animals exposed to 100% O2 at 1, 3, and 5 ATA. In 100% O2 at 1 ATA, control animals pretreated with saline showed a slightly elevated, but stable, brain PO2 compared with baseline levels during air breathing and little, if any, hyperoxic vasoconstriction. By comparison, animals receiving the PFC emulsion, after the initial responses to compression, exhibited a downward tendency in brain PO2 and rCBF (Figs. 1 and 2). In HBO2 at 3 ATA, rCBF and brain PO2 trended downward in both experimental groups, more so in PFC emulsion-treated animals, suggesting that increased PO2 is the ultimate driver of both effects. At 5 ATA, brain PO2 remained at a steady, although elevated, level for the first 30 min, and rCBF decreased signifi-

Table 3. Contributions of PFC emulsion (6 ml/kg) or saline infusion to blood O2 content Total CaO2, ml/dl HBO2, ATA

With saline infusion

With PFC emulsion infusion

⌬CaO2, ml/dl

1 3 5

23.4 27.9 32.4

25.3 34.1 42.9

1.9 6.2 10.5

⌬CaO2 represents change due to contribution of PFC emulsion.


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cantly in all four brain regions in control and PFC emulsion groups. After 30 min, however, a steeply rising hyperemia supervened. As expected, the most impressive changes occurred in the more highly perfused regions, the hypothalamus and striatum. At each level of hyperoxia, a specific pattern was observed that was roughly parallel between the control and PFC emulsion-treated rats, but generally changes were significantly greater in the latter. The sharp increase in brain PO2 at 5 ATA in PFC emulsion-treated animals (Fig. 5) would contribute to the shortening of seizure latency (Fig. 7). The downward trend in brain PO2 throughout the 60-min exposures at 1 and 3 ATA and for the first 30 min at 5 ATA (Figs. 2, 4, and 6) is associated with a concomitant decrease in rCBF that presumably represents an attempt to regulate cerebral O2 delivery. Our objective was to measure the effects of PFC emulsion under ordinary parameters of hyperbaric oxygenation, which could include small changes in PaCO2. Although not measured here, mild CO2 retention may occur in HBO2 (29). With constant ventilation, however, PaCO2 would rise by only 1 or 2 mmHg, not enough to alter cerebral vascular responses. In addition, CO2 is approximately five times more soluble than O2 in Oxycyte (26), so changes would be even smaller in PFC emulsion-treated animals. Moreover, no significant variations in PaCO2 have been reported in HBO2 at ⱕ3 ATA (10). It is also unlikely that altered cardiodynamics would have a significant effect on our results. Previously, we found that cardiac output fell by ⬃30% after 40 min of exposure to HBO2 at 6 ATA (8); however, such changes in cardiac output do not normally alter cerebral autoregulation (2). The increase in blood O2 content due to the PFC emulsion in circulatory equilibrium can be calculated for each pressure using the alveolar gas equation and the O2 content equation (Table 2). Thus, alveolar PO2 (PAO2) values for rats breathing 100% O2 at 1, 3, and 5 ATA without PFC emulsion are 673, 2,193, and 3,713 mmHg, respectively. Since PaO2 is normally ⬃90% of PAO2, the O2 content equation without infusion of PFC emulsion yields values shown in Tables 2 and 3. The published blood volume for rats of 63.7 ⫾ 6.2 ml/kg (14) and the body weight of the animals used in this study, 341 ⫾ 3 g, indicate a mean blood volume of 21.7 ml. Therefore, CaO2 at 1, 3, and 5 ATA is estimated to be 5.2, 6.1, and 7.12 ml, respectively. Since the pure PFC compound in the emulsion used here has a solubility for O2 of 7 ml/dl and comprises 60% of the emulsion (26), the 6 ml/kg dose contributes the following calculated volumes of O2 to each animal: 0.42 ml at 1 ATA, 1.37 ml at 3 ATA, and 2.32 ml at 5 ATA, which are normalized to milliliters per deciliter in Table 3. These values represent increases of 8.2%, 22.3%, and 32.6% compared with control animals. Therefore, in HBO2 at 5 ATA, infusion of PFC emulsion at 6 ml/kg raises the calculated total CaO2 in a 341-g rat to 9.4 ml (42.7 ml/dl), of which 4.7 ml (21.4 ml/dl) is bound to Hb, 2.4 ml (10.9 ml/dl) is dissolved in plasma, and 2.3 ml (10.6 ml/dl) is carried in the PFC emulsion, compared with a total of 7.1 ml (32.3 ml/dl) in control animals exposed to the same pressure. Since the normal cerebral metabolic rate for O2 is unaffected by infusion of PFC emulsion (16), the additional O2 overwhelms protective pathways, and CNS toxicity develops in all rats within 45 min of exposure. At the lower PFC emulsion dose (3 ml/kg), 80% of the animals experienced seizures before

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45 min. Since half of the control rats experienced seizures at 5 ATA, with a calculated total CaO2 of 32.4 ml/dl, this CaO2 represents the LD50 for rats exposed to O2 at this pressure for 45 min. Thus the additional 10.3 ml/dl contributed by the PFC emulsion at 6 ml/kg is enough to evoke seizures in 100% of the animals. Seizures were not observed in anesthetized rats pretreated with this PFC emulsion and exposed to HBO2 at 3 ATA for 60 min, suggesting that an increase in CaO2 of 6.2 ml/dl is tolerated by epileptogenic brain regions for exposures of this duration. However, in HBO2 at 5 ATA, Oxycyte clearly poses a significant risk for CNS O2 toxicity in conscious rats exposed to HBO2 for ⬎10 min. The implication for the future development of safe protocols is that the focus should be on administration of PFC emulsion after decompression, when PaO2 is not so elevated. Apart from prevention or mitigation of DCS and gas embolism, the ability of PFC emulsions to efficiently transport respiratory gases and tolerate long-term storage, combined with relatively low toxicity, make them potentially useful for treating severe hemorrhage, as well as vascular injuries to the brain and spinal cord. The emulsion particles are much smaller than erythrocytes and can therefore pass through constricted or damaged vessels. These agents are most effective in combination with elevated levels of inspired O2, but caution is advisable when they are used in HBO2, especially at ⱖ3 ATA. GRANTS This work was supported by the Naval Medical Research Institute through the Henry M. Jackson Foundation (subcontract under HU001-08-0003) and by the Office of Naval Research (N00014-11-1-0040). DISCLAIMER The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, or the US Government. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS I.T.D., R.T.M., B.W.A., and C.A.P. are responsible for conception and design of the research; I.T.D. performed the experiments; I.T.D. and B.W.A. analyzed the data; I.T.D., R.T.M., B.W.A., and C.A.P. interpreted the results of the experiments; I.T.D. and B.W.A. prepared the figures; I.T.D. and B.W.A. drafted the manuscript; I.T.D., R.T.M., B.W.A., and C.A.P. edited and revised the manuscript; I.T.D., R.T.M., B.W.A., and C.A.P. approved the final version of the manuscript. REFERENCES 1. Blogg S, Gennser M, Loveman GAM, Seddon FM, Thacker JC, White MG. The effect of breathing hyperoxic gas during simulated submarine escape on venous gas emboli and decompression illness. Undersea Hyperb Med 30: 163–174, 2003. 2. Bouma GJ, Muizelaar JP. Relationship between cardiac output and cerebral blood flow in patients with intact and with impaired autoregulation. J Neurosurg 73: 368 –374, 1990. 3. Castro CI, Briceno JC. Perfluorocarbon-based oxygen carriers: review of products and trials. Artif Organs 34: 622–634, 2010. 4. Dainer H, Nelson J, Brass K, Montcalm-Smith E, Mahon R. Short oxygen prebreathing and intravenous perfluorocarbon emulsion reduces morbidity and mortality in a swine saturation model of decompression sickness. J Appl Physiol 102: 1099 –1104, 2007. 5. Demchenko IT, Boso AE, O’Neill TJ, Bennett PB, Piantadosi CA. Nitric oxide and cerebral blood flow responses to hyperbaric oxygen. J Appl Physiol 88: 1381–1389, 2000.


Brain PO2, CBF, And O2 Toxicity in HBO2 With PFC 6. Demchenko IT, Boso AE, Whorton AR, Piantadosi CA. Nitric oxide production is enhanced in rat brain before oxygen-induced convulsions. Brain Res 917: 253–261, 2001. 7. Demchenko IT, Luchakov YI, Moskvin AN, Gutsaeva DR, Allen BW, Thalmann ED, Piantadosi CA. Cerebral blood flow and brain oxygenation in rats breathing oxygen under pressure. J Cereb Blood Flow Metab 25: 1288 –1300, 2005. 8. Demchenko IT, Moskvin AN, Krivchenko AI, Piantadosi CA, Allen BW. Nitric oxide-mediated central sympathetic excitation promotes CNS and pulmonary O2 toxicity. J Appl Physiol. First published March 22, 2012; doi:10.1152/japplphysiol.00902.2011. 9. Dromsky DM, Spiess BD, Fahlman A. Treatment of decompression sickness in swine with intravenous perfluorocarbon emulsion. Aviat Space Environ Med 75: 301–305, 2004. 10. Forkner IF, Piantadosi CA, Scafetta N, Moon RE. Hyperoxia-induced tissue hypoxia—a danger? Anesthesiology 106: 1051–1055, 2007. 11. Fraker CA, Mendez AJ, Stabler CL. Complementary methods for the determination of dissolved oxygen content in perfluorocarbon emulsions and other solutions. J Phys Chem B: 115: 10547–10552, 2011. 12. Fulton JF. Decompression Sickness, Caisson Sickness, Diver’s and Flier’s Bends and Related Syndromes. Philadelphia: Saunders, 1951, p. 437. 13. Henry F, Cook S. Effectiveness of pre-flight oxygen breathing in preventing decompression sickness. J Aviat Med 16: 350 –355, 1945. 14. LaRegina MC, Sharp PE. Important biological features. In: The Laboratory Rat. Orlando, FL: CRC, 1998, chapt. 1. 15. Latson GW, Flynn ET. Accelerated Decompression Using Oxygen for Submarine Rescue—Summary Report and Operational Guidance. Washington, DC: Dept. of the Navy, 2000. (Navy Experimental Diving Unit Technical Report 11-00) 16. Lee PA, Sylvia AL, Piantadosi CA. Effect of fluorocarbon-for-blood exchange on regional cerebral blood flow in rats. Am J Physiol Heart Circ Physiol 254: H719 –H726, 1988. 17. Lutz J, Herrmann G. Perfluorochemicals as a treatment of decompression sickness in rats. Pflügers Arch 401: 174 –177, 1984. 18. Lynch P, Krasner L, Vinciquerra T, Shaffer T. Effects of intravenous perfluorocarbon and oxygen breathing on acute decompression sickness in the hamster. Undersea Biomed Res 16: 275–281, 1989.

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Demchenko IT et al.

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19. Mahon R, Watanabe T, Wilson M, Auker C. Intravenous perfluorocarbon after onset of decompression sickness decreases mortality in 20-kg swine. Aviat Space Environ Med 81: 555–559, 2010. 20. Mahon RT, Dainer HM, Gibellato MG, Soutiere SE. Short oxygen prebreathe periods reduce or prevent severe decompression sickness in a 70-kg swine saturation model. J Appl Physiol 106: 1459 –1463, 2009. 21. Mahon RT, Dainer HM, Nelson JW. Decompression sickness in a swine model: isobaric denitrogenation and perfluorocarbon at depth. Aviat Space Environ Med 77: 8 –12, 2006. 22. McBarron JW. US prebreathe protocol. Acta Astronaut 32: 75–78, 1994. 23. Randsoe T, Hyldegaard O. Effect of oxygen breathing and perfluorocarbon emulsion treatment on air bubbles in adipose tissue during decompression sickness. J Appl Physiol 107: 1857–1863, 2009. 24. Riess JG. Oxygen carriers (“blood substitutes”) raison d’etre, chemistry, and some physiology. Blut ist ein ganz besondrer Saft1. Chem Rev 101: 2797–2920, 2001. 25. Skogland S, Stuhr LEB, Sundland H, Marstein S, Hope A. Increased oxygen before and during decompression reduces bubble formation in rats. Undersea Hyperb Med 30: 37–46, 2003. 26. Spiess BD. Perfluorocarbon emulsions as a promising technology: a review of tissue and vascular gas dynamics. J Appl Physiol 106: 1444 – 1452, 2009. 27. Spiess BD, McCarthy RJ, Tuman KJ, Woronowicz AW, Tool KA, Ivankovich AD. Treatment of decompression-sickness with a perfluorocarbon emulsion (FC-43). Undersea Biomed Res 15: 31–37, 1988. 28. Spiess BD, Zhu J, Pierce B, Weis R, Berger BE, Reses J, Smith CR, Ewbank B, Ward KR. Effects of perfluorocarbon infusion in an anesthetized swine decompression model. J Surg Res 153: 83–94, 2009. 29. Torbati D. Regional cerebral metabolic rate for glucose immediately following exposure to two atmospheres absolute oxygen in conscious rats. Neurosci Lett 55: 109 –112, 1985. 30. Yang ZJ, Price CD, Bosco G, Tucci M, El-Badri NS, Mangar D, Camporesi EM. The effect of isovolemic hemodilution with Oxycyte, a perfluorocarbon emulsion, on cerebral blood flow in rats. PLos One 3: e2010, 2008.
