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Ann Thorac Surg 2004;77:1656-1663
© 2004 The Society of Thoracic Surgeons
a Department of Cardiovascular Surgery Children's Hospital and Harvard Medical School, Boston, Massachusetts, USA
b Department of Biostatistics Children's Hospital and Harvard Medical School, Boston, Massachusetts, USA
c Department of Neurology Children's Hospital and Harvard Medical School, Boston, Massachusetts, USA
d Department of Radiology, Children's Hospital and Harvard Medical School, Boston, Massachusetts, USA
Accepted for publication October 10, 2003.
* Address reprint requests to Dr Jonas, Department of Cardiovascular Surgery, Children's Hospital, 300 Longwood Ave, Boston, MA, USA 02115
e-mail: richard.jonas{at}tch.harvard.edu
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Abstract |
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METHODS: Two experiments were conducted and analyzed separately. In each experiment, 10 piglets were randomized to two different groups, one with a total blood prime yielding a high hematocrit (25% or 30%), and the other with a crystalloid prime resulting in a low hematocrit (10% or 15%). Animals were cooled with pH-stat strategy at full flow (100 or 150 mL · kg–1 · min–1) to a nasopharyngeal temperature of 15°C, a period of low flow (50 mL · kg–1 · min–1) preceding deep hypothermic circulatory arrest (45 or 60 minutes), and a period of rewarming at full flow. Cerebral blood flow was measured at the beginning of CPB, at the end of cooling, at the end of low flow, 5 minutes after the start of rewarming, and at the end of rewarming by injection of radioactive microspheres.
RESULTS: Mean arterial pressure was significantly greater with higher hematocrit at each time point (p< 0.05). Cerebral blood flow and the cerebral metabolic rate of oxygen decreased during cooling and further during low flow bypass but were significantly greater with lower hematocrit during mild hypothermia and at the end of rewarming (p< 0.05).
CONCLUSIONS: Hemodilution is associated with decreased perfusion pressure, increased CBF and increased the cerebral metabolic rate of oxygen during hypothermic CPB.
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Introduction |
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An alternative explanation, however, which forms the hypothesis for the current study, is that even in settings of a fixed "cardiac output" provided by a CPB pump, CBF is increased under hemodilution conditions. However, at severe degrees of hemodilution the reduced oxygen-carrying capacity of blood may result in inadequate cerebral oxygen delivery.
Two sets of experiments were undertaken. On analysis of the data from the first set of animals, we were concerned that an artifact in measurement of CBF or the cerebral metabolic rate of oxygen (CMRO2) had resulted in the unexpected findings. The second set of experiments used a modified method for great vessel cannulation and microsphere measurement of CBF as well as slightly different flows, hematocrit levels, and temperatures.
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Material and methods |
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The chest was opened by a median sternotomy. After systemic heparinization (300 IU/kg), an 8F arterial cannula (DLP, Medtronic Inc, Grand Rapids, MI) and 28F venous cannula (Bard, Inc, USCI Division, Billerica, MA) were inserted into the right femoral artery (experiment 1), ascending aorta (experiment 2) and the right atrium, respectively. A venous line was placed as high as possible in the jugular vein through the superior vena cava to measure the jugular venous saturation during the study. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and the Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institute of Health (National Institutes of Health Publication No. 86-23, revised 1985).
Cardiopulmonary bypass technique
The CPB circuit consisted of a roller pump, membrane oxygenator (Minimax, Medtronic Inc, Anaheim, CA), and sterile tubing with a 40-µm arterial filter (Olson Medical Sales Inc, Ashland, MA). The pump prime consisted of a balanced electrolyte solution Plasmalyte A (Baxter, Deerfield, IL) and freshly drawn heparinized blood to achieve the desired hematocrit (see study groups below). Fresh whole blood or crystalloid solution was transfused into the pump as required to keep the hematocrit at the target level. Methylprednisolone (30 mg/kg), furosemide (0.25 mg/kg), sodium bicarbonate (10 mL), fentanyl (50 µg/kg), and pancuronium (0.5 mg/kg) were added to the prime. Full bypass flow was set at 150 mL · kg–1 · min–1 in experiment 1 and 100 mL · kg–1 · min–1 in experiment 2. Ventilation was stopped after the onset of CPB. A venting cannula was inserted into the left ventricle and the ascending aorta was clamped to prevent residual forward blood flow from the heart.
In experiment 1, after reaching mild hypothermia and maintaining esophageal temperature at 34°C for 10 minutes, experimental conditions were kept constant to allow measurement of CBF. These and the following measurements took 15 minutes each. The piglet was then cooled to an esophageal temperature of 15°C for more than 30 minutes applying the pH-stat strategy, ie, addition of CO2 to maintain the temperature corrected pH at 7.40 [4, 5] followed by a second measurement period. Low flow bypass was then initiated at a flow rate of 50 mL · kg–1 · min–1. After 15 minutes of low flow, the third measurement period was started. Following 45 minutes of deep hypothermic circulatory arrest (DHCA), furosemide (0.25 mg/kg), sodium bicarbonate (10 mL), and mannitol (0.5 g/kg) were administered into the pump and reperfusion was initiated. Within 5 minutes, the temperature of the piglet had increased to 20°C and was controlled at this level for the fourth measurement period. Subsequently, the piglets were fully rewarmed more than 40 minutes and then the last measurements were taken.
In experiment 2, animals were perfused for 10 minutes to achieve steady state at normothermia (37°C), and the experimental conditions were kept constant to allow for measurement of CBF. These and the following measurements took 10 minutes each. Animals were then cooled to an esophageal temperature of 15°C for more than 30 minutes applying the pH-stat strategy followed by a second measurement period. Low flow bypass was then initiated at a flow rate of 30 mL · kg–1 · min–1. After 10 minutes of low flow, the third measurement period was started. Then animals underwent DHCA for 60 minutes. Before reperfusion, furosemide (0.25 mg/kg), sodium bicarbonate (10 mL), and mannitol (0.5 g/kg) were administered into the pump. Reperfusion was begun at 100 mL · kg–1 · min–1. Within 5 minutes, the temperature of the piglet had increased to 20°C and was controlled at this level for the fourth measurement period. Subsequently, the piglets were fully rewarmed for more than 30 minutes and then the last measurement was performed at 37°C.
Data collection
Blood flow measurements
Regional blood flow to the brain was measured by microspheres labeled with one of the following radioactive nuclides: 141Ce, 51Cr, 103Ru, 95Nb, or 46Sc suspended in 2 mL of 0.9% saline followed by 2 mL of 0.9% saline injected into a side port on the arterial tubing. In experiment 1, the side port was upstream to the arterial filter (40-µm mesh) and 80 cm from the tip of the arterial cannula to ensure complete mixing. In experiment 2, the side port was downstream to the filter and 1 m from the tip of the arterial cannula. Each microsphere injection consisted of approximately 2.5 x 106 microspheres. A measured quantity of blood (approximately 5 mL) as reference was withdrawn at a constant rate by a syringe pump (model 55:1143, Harvard Apparatus) from the arterial line placed in left femoral artery. Arterial blood was withdrawn before, during, and after the microsphere injection for a total of 90 seconds. At the end of each experiment, the brain was removed and weighed. Three samples were taken from the left and right frontal cortex. Additional samples were taken from the left and right parietal and occipital cortex, the left and right cerebellum, pons, and medulla oblongata. Radioactivity was counted with a gamma counter (Cobra II Autogamma, Packard Instrument, IL) with a spillover correction between the nuclides. The reference blood was also analyzed for radioactivity. Regional blood flow calculated from the rate of withdrawal of the reference blood and the ratio of the radioactivity of the brain to the reference blood. Average CBF was derived by adding the average regional blood flows and dividing by number of regions.
Metabolic measurements
Blood gas tensions and pH, hematocrit, plasma glucose, and lactate levels were measured in arterial blood and blood withdrawn from the jugular bulb before CPB and before each microsphere injection. Measurements including hemoglobin, saturation, and oxygen content were performed by a blood gas analyzer (Stat Profile 9, Nova Biomedical, Waltham, MA). Cerebral oxygen delivery (CDO2), cerebral metabolic rate for oxygen (CMRO2), and oxygen extraction ratio (OER) were calculated by standard formulas [6] and listed in the Appendix.
Hematocrit study groups and power analysis
In experiment 1, a hematocrit of 15% was specified for low hematocrit and 25% for high hematocrit during CPB. In experiment 2, a low hematocrit was specified as 10% and a high hematocrit was 30% or freshly drawn heparinized blood to achieve a high hematocrit of 30%. Because the protocols and hematocrit study groups were not identical between the two sets of experiments, we chose to analyze the experiments separately. The sample size of 10 piglets equally randomized to the two hematocrit study groups provided 80% statistical power (beta error = 0.20) to detect differences based on a desired effect size of 1.75 in each of the CBF and metabolic variables in each experiment using analysis of variance (ANOVA) with repeated measures with a Bonferroni corrected significance level of 0.01 (0.05 divided by 5) to account for the multiple time points of the study (version 4.0, nQuery Advisor, Statistical Solutions, Boston, MA) [7].
Statistical analysis
Data are represented as means ± SD. After verifying the assumption of normality by the Kolmogorov-Smirnov test [8], continuous variables including hematocrit level, arterial oxygen pressure (PO2), arterial carbon dioxide pressure (PCO2), jugular venous pH, esophageal and rectal temperature, and mean arterial pressure (MAP) were compared at each of five time points (mild hypothermia, end of cooling, after 15 minutes of low flow, 5 minutes after rewarming, end of rewarming) between the two hematocrit groups in each experiment using unpaired Student t tests. Two-way repeated-measures ANOVA was used to test the interaction between experiment and hematocrit level for the four main outcome variables (CBF, CDO2, CMRO2, and OER) and to assess changes across the time points and between hematocrit study groups [9]. Statistical significance was established using a two-tailed value of p< 0.05 with post hoc Bonferroni adjustment. Multivariate linear regression analysis was applied to ascertain whether the differences between low and high hematocrit level was independent of differences in MAP. Analysis of the data were performed with the SPSS software package (version 11.0, SPSS Inc, Chicago, IL).
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The comparisons between hematocrit groups within each experiment for CBF, CDO2, CMRO2, and OER are presented in Table 2. Measures of cerebral metabolism were significantly different between low and high hematocrit study groups within each experiment. Higher CBF was observed in the low hematocrit group at the beginning of cooling (ie, mild hypothermia) and at the end of rewarming for experiments 1 and 2 (Fig 2). In addition, a higher CBF was demonstrated among the animals in the low hematocrit group at the end of cooling and during low flow bypass in experiment 2. In both sets of experiments, CBF decreased sharply between mild hypothermia and the end of cooling and continued to decline throughout low flow and increase during the rewarming phase. This time-related pattern for the change in CBF was significant for both experiments (p< 0.01, F test for time effect).
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Comment |
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Consistent with the results of this study, several previous studies from our laboratory [3] and preliminary results of a prospective, randomized clinical trial in infants [10] have demonstrated beneficial effects of a high hematocrit (30%) during CPB with and without DHCA. In a piglet survival model described by Shin'oka and associates [11], both functional and structural outcome measures were improved with a higher hematocrit. The mechanism appeared to be related, in part, to inadequate reduced oxygen availability with a low hematocrit as determined by near-infrared spectroscopy [11]. Magnetic resonance spectroscopy demonstrated a decrease in high-energy phosphate levels and a drop in intracellular pH, particularly in the early phase of cooling with a low hematocrit (10%).
Blood pressure versus pump flow as determinants of cerebral blood flow
Other groups have demonstrated previously that with pH-stat management, as used in the current study (ie, addition of CO2 to correct for the alkaline shift associated with hypothermia), CBF autoregulation is impaired leading to pressure and flow-dependent perfusion of the brain [12]. Therefore, the higher arterial pressure associated with higher hematocrit could per se improve neurologic outcome. Indeed clinical reports on adult cardiac surgery have indicated that higher arterial pressures (MAP > 70 mm Hg) are beneficial for neurologic outcome [13], although this population likely had both carotid and cerebrovascular disease. However, in both of our experiments, despite the use of pH-stat, CBF was independent of MAP over a wide range of perfusion pressures, ie, autoregulation was intact, which has been well described for this range with 
-stat management [12, 14]. In contrast to blood pressure, pump flow in our study had a significant influence on CBF.
Few previous studies have attempted to differentiate between the effects on CBF of CPB perfusion flow rate and blood pressure in the setting of pH-stat management. Previous researchers have used phenylephrine and sodium nitroprusside to vary MAP while maintaining a constant pump flow [15, 16]. An increase of CBF with greater perfusion pressure was demonstrated, although the specific pharmacologic effects of the vasoactive drugs used in that study on cerebral resistance vessels are unknown. In another study, investigators manipulated perfusion pressure in the carotid artery independent of the perfusion flow rate by obstructing the descending aorta [17]. They found a highly significant correlation between CBF and blood pressure but not flow, but conceded that constriction of the aorta during low flow in order to increase blood pressure in the carotid artery may redirect blood flow to the brain. The relevance to clinical practice remains unclear. In contrast, others have demonstrated in clinical studies a dependency of CBF on perfusion flow but not pressure [18, 19].
Hematocrit and cerebral blood flow
In the current study, hemodilution generally increased CBF though not significantly early rewarming after DHCA. Microcirculatory dysfunction of cerebral vessels after prolonged DHCA has been described previously by others and may account for the fact that CBF measurements after DHCA were lower than at baseline, although a hyperemic response after ischemia might be expected [20]. In our own studies using intravital microscopy, we have observed reduced flow in the early reperfusion phase with a low relative to high hematocrit, which we have attributed to hypoxic endothelial injury resulting in impaired NO synthesis [21].
The influence of hematocrit on CBF has been studied most commonly in the past in a non-CPB setting [22]. An increase in CBF with decreasing hematocrit has been found in all studies but cardiac output was not controlled and therefore almost certainly increased significantly in all such studies; however, in some studies, oxygen delivery declined because of the decreased oxygen-carrying capacity of dilute blood [22]. In an important study by Cook and colleagues [23] using a canine model of CPB it was found that cerebral oxygen delivery was maintained with hemodilution. However, a significant difference in Cook's study from our own was that pump flow rate was increased in their study to compensate for the decrease in perfusion pressure resulting from hemodilution.
Hematocrit and cerebral metabolism
Probably the most surprising finding of our study was that CMRO2 was higher in the low hematocrit group at all temperatures. It was this finding that led us to completely repeat the study using aortic rather than femoral arterial cannulation. We were concerned that the microsphere assessment of CBF may have been unreliable with femoral arterial cannulation because the reference blood was drawn from the other femoral artery. Also in experiment 1, the microsphere injections were made upstream to an arterial filter. Although the mesh size of the filter should not have trapped microspheres, we speculated that this possibility could have artificially elevated our assessment of CBF and therefore CMRO2. However, the results obtained in experiment 2 by a completely new laboratory team and with aortic rather than femoral cannulation were similar to those obtained in experiment 1.
Previous piglet studies from our laboratory have demonstrated that during cooling lower hematocrit is associated with lower levels of cerebral high-energy phosphates (ATP and phosphocreatine) and intracellular acidosis [3]. In the current study, lower SjO2 and arterial pH were also noted. These data also suggested to us that cerebral oxygen demands were not being met during CPB. However, the present study indicated that part of the problem may be an increasing demand for oxygen with low arterial oxygen content despite maintenance of oxygen delivery by markedly increased CBF. Perhaps more oxygen is needed to counteract acidosis due to the lower pH-buffering capacity of the dilute blood. Other possible mechanisms could be an increase in substrate transport energy requirements with more dilute blood or perhaps some energy-requiring aspect of the markedly increased CBF itself. In theory, the onset of anaerobic metabolism should reduce CMRO2 but this may be a late phenomenon.
A recent clinical trial in infants has demonstrated a deleterious effect of hemodilution [10] and some clinical studies in adult cardiac surgery have correlated poor neurologic outcome with lower intraoperative hemoglobin levels [24, 25]. In addition to the risk of hypoxic injury suggested by this study and in our previous studies, excessive CBF due to hemodilution may possibly increase the embolic load to the brain similar to the effect of pH-stat management in adults. Another possible explanation for the neurologic damage observed in adults with hemodilution is increased edema formation.
Limitations of the study
The lowest mean arterial blood pressure in our study was 20 mm Hg in the low hematocrit group during low-flow bypass. Cerebral venous blood was drawn from the jugular bulb and not from the sagittal sinus. Therefore, we cannot exclude the possibility that the venous sample was contaminated by extracerebral blood sources leading to an underestimation of cerebral oxygen metabolism. However, sampling from the jugular bulb to represent brain venous blood is the gold standard in humans [6].
There is no doubt that CBF autoregulation may be altered by age and coexistent disease such as diabetes mellitus, hypertension, or atherosclerotic cerebrovascular disease [6]. It may not be appropriate to extrapolate our results from 4-week old piglets to an adult pig population or to humans. Nevertheless, the lower perfusion pressure that results from hemodilution could only exacerbate a watershed injury from hypoperfusion secondary to carotid or cerebrovascular disease. Furthermore, the greater CBF with hemodilution could only increase the microembolic load if atheromatous debris is dislodged by the manipulations of CPB.
In summary, the results of this study call into question the common clinical practice of moderate-to-severe hemodilution during CPB in adults and children. In children there is a risk of hypoxic injury because of the increase in CMRO2, which appears to result from hemodilution. Adults are at an increased risk with hemodilution secondary to both hypoxic and microembolic injury. Perhaps both of these mechanisms may explain the disturbingly high incidence of permanent cognitive dysfunction observed in adults after CPB [26].
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Appendix |
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This article has been cited by other articles:
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  J. K. Shoemaker Hemodilution Impairs Cerebral Autoregulation, Demonstrating the Complexity of Integrative Physiology Anesth. Analg., November 1, 2007; 105(5): 1179 - 1181. [Full Text] [PDF]
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 J. C. Halstead, M. Wurm, D. M. Meier, N. Zhang, D. Spielvogel, D. Weisz, C. Bodian, and R. B. Griepp Avoidance of hemodilution during selective cerebral perfusion enhances neurobehavioral outcome in a survival porcine model Eur. J. Cardiothorac. Surg., September 1, 2007; 32(3): 514 - 520. [Abstract] [Full Text] [PDF]
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