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J Thorac Cardiovasc Surg 1999;117:1166-1171
© 1999 Mosby, Inc.

SURGERY FOR CONGENITAL HEART DISEASE

OXYGENATION STRATEGY AND NEUROLOGIC DAMAGE AFTER DEEP HYPOTHERMIC CIRCULATORY ARREST. I. GASEOUS MICROEMBOLI

From the Department of Cardiac Surgery, Children's Hospital, and the Department of Surgery, Harvard Medical School, Boston, Mass.

Supported by a Habilitandenstipendium of the Deutsche Forschungsgemeinschaft NO344/1-1 (G.N.).

Received for publication Aug 28, 1998. Revisions requested Oct 30, 1998. Revisions received Feb 2, 1999. Accepted for publication Feb 19, 1999. Address for reprints: Richard A. Jonas, MD, Department of Cardiac Surgery, Children's Hospital, 300 Longwood Ave, Boston, MA 02115.

	Abstract

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Objectives: Recent studies suggest that myocardial reperfusion injury is exacerbated by free radicals when pure oxygen is used during cardiopulmonary bypass. Partial replacement of the oxygenator gas mixture with nitrogen, however, such as has already been adopted clinically in many centers, could increase the risk of gaseous nitrogen microembolus formation and therefore of brain damage because of the low solubility of nitrogen, particularly under conditions of hypothermia.
Methods: Ten 7- to 10-kg piglets were cooled for 30 minutes to 15°C on cardiopulmonary bypass and then rewarmed for 40 minutes to 37°C. In 5 piglets cardiopulmonary bypass was normoxic and in 5 it was hyperoxic. In each group 3 bubble oxygenators without arterial filters and 2 membrane oxygenators with filters were used. Cerebral microemboli were monitored continuously by carotid Doppler ultrasonography (8 MHz) and intermittently by fluorescence retinography.
Results: Embolus count was greater with lower rectal temperature (P < .001), use of a bubble oxygenator (P < .001), and lower oxygen concentration (P = .021) but was not affected by the temperature gradient between blood and body during cooling or rewarming.
Conclusions: Gaseous microemboli are increased with normoxic perfusion, but this is only important if a bubble oxygenator without a filter is used. (J Thorac Cardiovasc Surg 1999; 117:1166-71)

	Introduction

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In the early years of oxygenator technology pure oxygen was used as a safety measure to ensure sufficient blood oxygenation. Recent experimental and clinical data, however, suggest that the hyperoxygenation achieved with modern oxygenators during reperfusion aggravates ischemia-reperfusion injury through generation of oxygen free radicals, particularly in the heart. ^1-3 Normoxic cardiopulmonary bypass (CPB) has therefore been advocated and is now part of clinical practice in many centers. In normoxic CPB oxygen is replaced by nitrogen. Because nitrogen is less soluble than oxygen, we speculated that normoxic CPB might increase gaseous microemboli ⁴ and reduce brain oxygen supply, with the potential to overwhelm any beneficial effects against reoxygenation injury. The aim of this study was therefore to investigate the effects of normoxic and of hyperoxic CPB on neurologic outcome in a piglet model of prolonged deep hypothermic circulatory arrest. In the first phase of the study the influence of normoxia on the number of gaseous emboli passing to the brain was investigated. Cerebral oxygenation and brain injury were assessed in the second phase.

	Methods

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Experimental preparation
Yorkshire piglets weighing 7.2 to 10.2 kg (mean 8.6 kg) were anesthetized with 15 to 20 mg/kg intramuscularly injected ketamine and intubated with a 5-mm cuffed endotracheal tube. The animals were ventilated with a pressure-controlled ventilator (Healthdyne model 105; Respironics, Inc, Pittsburgh, Pa) with a peak inspiratory pressure of 25 cm H₂O and an inspired oxygen fraction (FIO₂) of 0.3 (normoxic group) or 1.0 (hyperoxic group) at a rate of 12 breaths/min. After an intravenous bolus of 25 µg/kg fentanyl and 0.5 mg/kg pancuronium bromide, anesthesia was maintained by continuous infusion of 20 to 40 µg · kg^–1 · h^–1 fentanyl and 0.2 mg · kg ^–1 · h^–1 pancuronium bromide throughout the entire experiment. Esophageal and rectal temperatures were recorded continuously. The left femoral artery was cannulated for arterial blood pressure monitoring and blood gas sampling. After exposure of the left internal jugular vein, a catheter was placed in the jugular bulb to allow blood withdrawal. The chest was opened by a median sternotomy. After systemic heparinization (300 IU/kg heparin), a 10F arterial cannula (Medtronic DLP, Grand Rapids, Mich) and 24F venous cannula (USCI Division, C.R. Bard Inc, Billerica, Mass) were inserted into the ascending aorta and the right atrium, respectively.

All animals received humane care in compliance with the "Principles of Laboratory Animal Care," formulated for the National Society for Medical Research, and the "Guide for the Care and Use of Laboratory Animals," prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985).

CPB technique. The CPB circuit consisted of a roller pump (Cardiovascular Instrument Corp, Wakefield, Mass), sterile tubing (OLSON Medical Sales Inc, Ashland, Mass), and either a membrane oxygenator (VPCML plus; COBE Cardiovascular, Inc, Arvada, Colo) with 40-µm arterial filter (pediatric extracorporeal blood filter; Pall Biomedical, Inc, Fajardo, PR) or a bubble oxygenator (Bentley Bio2; Baxter Healthcare Corporation CardioVascular Group, Irvine, Calif) without any arterial filter system. The pump prime consisted of a balanced electrolyte solution and freshly drawn heparinized blood to achieve a hematocrit between 20% and 25% during CPB. The prime was dosed with 25 mg/kg cefazolin sodium, 30 mg/kg methylprednisolone sodium succinate, 0.25 µg/kg furosemide, and 10 mL sodium bicarbonate. Full bypass flow was set at 100 mL · kg^–1 · min^–1. The animal was immediately cooled to an esophageal temperature of 15°C during 30 minutes according to alpha-stat strategy, which was selected to avoid the complexity of blending 3 gases in the laboratory setting and because of its current widespread clinical use. Ventilation was stopped after the establishment of CPB. After 30 minutes of cooling, 0.25 mg/kg furosemide, 0.5 g/kg mannitol, and 10 mL sodium bicarbonate were administered into the pump. The animal was then rewarmed to a temperature of 37°C during 40 minutes. The heart was defibrillated as necessary at 25°C. Fresh whole blood was transfused into the pump as required to increase the hematocrit to a range of 20% to 25% during rewarming.

Data collection
Doppler ultrasonography. An 8-MHz continuous-wave probe placed directly over the right common carotid artery was connected to a TCD monitor (MedaSonics CDS; Nicolet Biomedical Inc, Madison, Wis) and both the acoustic and video signals were recorded continuously on tape during CPB. Criteria for emboli were a sudden and sharp increase of the visual signal of more than 30% (Fig. 1) and a high pitched sound similar to chirping or whistling. ⁴ Quantification of emboli by this method is limited to approximately 150 emboli/min, because the signals cannot be discriminated at higher embolic rates. Higher counts of emboli were therefore arbitrarily recorded as 150 emboli/min, as previously suggested, ⁴ although embolus counts might have exceeded 500 emboli/min at low temperatures.

View larger version (164K): [in this window] [in a new window]   Fig. 1 Monitoring of emboli during CPB by Doppler ultrasonography. An 8-MHz continuous-wave Doppler flow probe was placed over right common carotid artery. The x-axis shows time frame of 6 seconds. A, Typical flow pattern during CPB in piglet operated on with membrane oxygenator and 40-µm arterial filter. B-D, Increasing numbers of emboli during cooling in pig operated on with bubble oxygenator without filter. Emboli are characterized visually by a sharp increase of more than 30% of signal. Blood foam containing massive amounts of gas bubbles were injected at end of experiment. E, Onset of this massive air embolism. F, Peak of embolism.

Blood gas values and biochemical analyses. Arterial and venous (jugular bulb) blood gas values, including electrolyte, glucose, and lactate levels, were measured (Nova 900; Nova Biomedical, Waltham, Mass) at baseline, 5 minutes after start of CPB, every 10 minutes during CPB, 30 minutes after the end of CPB, and at the end of the experiment.

Experimental groups
In 5 piglets CPB was normoxic (FIO ₂ 20%-40%) and in 5 it was hyperoxic (FIO ₂ 100%). In each group bubble oxygenators without arterial filters were used for 3 animals and membrane oxygenators with 40-µm filters were used for 2 animals. At the end of the experiment in each group gas bubbles were directly injected into the aortic cannula to validate the methods used for embolus detection.

Statistical analysis
All results were expressed as mean and standard error of the mean. The Student t test was used to compare absolute quantitative values between the groups. To assess differences between time points within groups the paired t test was used, and if applicable the results were corrected for multiple testing according to the method of Bonferroni. Correlation coefficients were calculated according to the method of Pearson for continuous data; Spearman coefficients were calculated for discrete data. Multiple stepwise linear regression was performed by the backward elimination method with an entry criterion of P < .10 and a criterion of P < .05 for being kept in the model. Statistical analyses were facilitated with the help of SPSS statistical software (version 7.0 for Windows; SPSS Inc, Chicago, Ill).

	Results

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Doppler ultrasonography
Embolus counts increased during cooling (Fig. 1; Table I) and decreased during rewarming with both types of oxygenator. They were significantly lower at every time point during CPB with membrane oxygenators with filters than with bubble oxygenators without filters (P < .001).

Retinography
Retinography was feasible in all animals and demonstrated delayed filling of retinal vessels 5 minutes after rewarming. Gaseous emboli could not be detected retrospectively by discontinuous retinography (Fig. 2). Retinography demonstrated only temporary obstruction of retinal vessels after injection of massive air bubbles, with complete reopening within 2 minutes (Fig. 2, E-G).

View larger version (119K): [in this window] [in a new window]   Fig. 2 Retinography during CPB. Fundus of pig is shown by conventional retinography (A). Fluorescent retinography of left eye was performed by venous injection of dye before aortic cannulation (B), 5 minutes after start of rewarming (C), and at end of rewarming (D). At end of experiment blood foam mixed with dye was injected into aortic cannula. E, Fundus before injection. F, Immediately after injection retinal vessels become obstructed. One minute later (G) all vessels opened up again.

	Discussion

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Further support for the concept that the gaseous microemboli detected in the carotid artery were not generated by gas coming out of solution because of rapid warming, either of the cold oxygenator blood during cooling or of the pig's cold arterial blood during rewarming, is provided by the observation that simply replacing a bubble oxygenator with a membrane oxygenator resulted in almost complete elimination of the microemboli. Perhaps gas coming out of solution could be important more distally in the arterial tree. The transducer used to detect microemboli in the common carotid artery was less than 10 cm from the arterial cannulation site in the ascending aorta. Assuming a flow velocity of more than 30 cm/s in the ascending aorta, bovine trunk, and carotid artery, warming and bubble formation would have to take place within 0.3 second. ⁶ There was no indication by retinography, however, that gaseous microemboli had an important role in permanently occluding small vessels more distally.

Retinography in fact proved to be remarkably insensitive in detecting gaseous microemboli. We were fortunate to be able to work with persons highly skilled in this technique and with state-of-the-art equipment from the Joslin Diabetes Center. We were therefore surprised when we were unable to demonstrate embolic vascular occlusions despite recording carotid microembolus counts greater than 500 emboli/min. This observation led us to conclude each study with the injection of a large amount of air directly into the aortic cannula, which revealed that the gaseous emboli caused obstruction of the retinal vessels for only a few seconds. Presumably the longer lasting occlusions of retinal vessels seen in previously reported clinical studies ⁷ were caused by fat or by atherosclerotic particulate emboli. This is not to say that transient gaseous emboli are benign. Previous studies have shown that they can cause endothelial dysfunction, with leukocyte and platelet adhesion leading to eventual capillary obstruction. ⁸ In support of this observation is the correlation that we observed between lactate level and the carotid artery embolus count.

The brain is subjected to several possible sources of injury during correction of congenital cardiac anomalies. During circulatory arrest ischemia itself and the subsequent reperfusion can lead to brain injury caused by calcium influx; the neurotoxicity of excitatory neurotransmitters such as dopamine, glutamate, and aspartate; and lipid peroxidation caused by free radicals. ⁹ High oxygen pressures during reperfusion may aggravate reperfusion injury by increased formation of free radicals. For example, nitric oxide transformation to the cytotoxic peroxynitrite or hydroxyl radicals is more likely in the presence of high oxygen pressures. ¹⁰ CPB with F IO₂ of 1.0 results in oxygen pressures of 400 mm Hg and greater. Ihnken and associates ¹ have reported higher lipid oxygenation, increased nitric oxide formation, and worse cardiac contractility after hypoxia and ischemia-reperfusion of immature canine hearts with hyperoxic management of CPB than with normoxic management. Laboratory studies elsewhere also have demonstrated beneficial effects for the brain of normoxic reperfusion with respect to hyperoxic reperfusion after ischemia. Reoxygenation with FIO ₂ 0.2 reduced oxygen radical–mediated injury of neurons and resulted in better recovery of dopamine metabolism than did reoxygenation with 100% oxygen after hypoxia ^11,12 and ischemia ¹³ in pigs and rabbits. Graded postischemic reperfusion by increasing arterial FIO₂ levels during 30 minutes from 0.1 to 0.21 showed further protective effects for neurons as long as 2 days after the operation. ¹⁴

Clinical studies comparing hyperoxic and normoxic management of CPB in cyanotic children have also revealed higher oxygen free radical damage after reperfusion, as assessed by products of lipid peroxidation. ^2,3 However, these studies have not assessed the potential for greater hypoxic injury or injury related to increased gaseous microemboli. Many cardiac surgery centers have nevertheless changed during the last 5 years from hyperoxic to normoxic management of CPB, even though direct clinical benefit has not been demonstrated by any prospective trials.

If membrane oxygenators are used in conjunction with arterial line filters, normoxic oxygenation during CPB does not increase the number of gaseous cerebral microemboli. Bubble oxygenators without an arterial line filter should not be used for normoxic CPB. Traditional concepts regarding temperature gradients and risk of gaseous microemboli should be reexamined.

	Acknowledgments

 
We thank Mark Cioffi, Pascal Gebeyan, and Christine Rader, from the Department of Cardiac Surgery, and Gene Walter, from the Department of Neurology, for technical support during the operations. Assistance with data analysis by David Zuraowski, PhD, statistician, Department of Biostatistics, and Frank Perron, PhD, Joslin Diabetes Center, is greatly appreciated. We also acknowledge the assistance of Allen Clermont, MS, and Sven Bursell, PhD, from the Joslin Diabetes Center for assistance with retinography and Laverne Gugino, MD, for his assistance with carotid Doppler studies. We thank Laura Young for preparation of the manuscript.

	References

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