Asian Cardiovasc Thorac Ann 2002;10:223-227
© 2002 Asia Publishing EXchange Pte Ltd
Cerebral Metabolism of Nitric Oxide During Retrograde Cerebral Perfusion
Katsuhito Ueno, MD,
Shinichi Takamoto, MD,
Takeshi Miyairi, MD,
Tetsuro Morota, MD,
Ko Shibata, MD,
Arata Murakami, MD,
Yutaka Kotsuka, MD
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Department of Cardiothoracic Surgery Faculty of Medicine University of Tokyo Tokyo, Japan
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For reprint information contact: Katsuhito Ueno, MD Tel: 81 3 3815 5411 Ext. 33321 Fax: 81 3 5684 3989 email: kueno-tky{at}umin.ac.jp Department of Cardiothoracic Surgery, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
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ABSTRACT
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The aim of this study was to determine whether alpha- or pH-stat protects the brain during deep hypothermic retrograde cerebral perfusion. Fifteen anesthetized dogs on cardiopulmonary bypass were cooled to 18°C under alpha-stat and underwent retrograde cerebral perfusion for 90 minutes under alpha-stat or pH-stat, or underwent antegrade cardiopulmonary bypass under alpha-stat as the control. Cerebral blood flow of the cortex was monitored and serial analyses of blood gases and total nitric oxide oxidation products made. Cerebral blood flow and cerebral metabolic rate for oxygen were significantly higher and plasma levels of nitric oxide oxidation products in the outflow from the brain were significantly lower in retrograde cerebral perfusion under pH-stat than under alpha-stat. This study shows that reduced levels of nitric oxide oxidation products may protect against neuronal damage induced by nitric oxide and that increased cerebral blood flow under pH-stat may lead to a reduction of nitric oxide oxidation products. Under retrograde cerebral perfusion, pH-stat is thus better than alpha-stat for protecting the brain.
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INTRODUCTION
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Many types of physiological intervention have been assessed in the attempt to improve the outcome of retrograde cerebral perfusion (RCP). The effects of hemodilution, oxygenation, and perfusion flow and pressure have all been studied, but there is still no agreement on the most appropriate management of arterial carbon dioxide (CO2) and whether alpha-stat or pH-stat gives better results. It has been reported that plasma levels of total nitric oxide oxidation products (NOx) increased after transient brain ischemia,1 that glutamate excitotoxicity plays a role in neurological injury with nitric oxide (NO) as a major mediator of N-methyl-D-aspartate (NMDA) receptor–mediated neuronal death,2 and that glutamate receptor agonists cause a dose-dependent increase in brain plasma NOx levels.3
The present study was undertaken to study optimal CO2 control in the perfusate during RCP in terms of cortical cerebral blood flow (CBF) and cerebral metabolism of oxygen and NO.
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MATERIALS AND METHODS
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Fifteen adult mongrel dogs (mean weight, 25.1 ± 1.1 kg) were used in this study. All received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institute of Health (NIH publication 85-23). The dogs were premedicated with ketamine hydrochloride (15 mgákg–1) intramuscularly and pentobarbiturate (35 mgákg–1) intravenously. Anesthesia was maintained with an additional dose of pentobarbiturate (0.5 mgákg–1) as required. The dosage of pentobarbiturate was equivalent between the study groups.
Under general anesthesia, the dura mater and superior sagittal sinus were exposed by craniotomy. The probe of a laser Doppler flowmeter (ALF21; Advance Co., Tokyo, Japan) was placed on the cortical surface of the left temporal lobe to monitor CBF of the cortex, and a brain thermometer was inserted into the cortical surface of the right temporal lobe. In addition, an indwelling needle was inserted into the exposed superior sagittal sinus for pressure monitoring.
A median sternotomy was then performed. Heparin sodium (300 Uákg–1) was administered, and a 14F arterial cannula was inserted into the right femoral artery. Venous drainage from the cardiopulmonary bypass (CPB) circuit went through a 32F right atrial cannula. CPB was then instituted. Blood was circulated by a roller pump through a combined heat exchanger–oxygenator [Capiox SX (M); Terumo Co., Tokyo, Japan] with a cardiotomy reservoir primed with lactated Ringer’s solution, sodium bicarbonate, and heparin sodium. Hemodynamic and electrophysiological monitoring was carried out. Brain temperature was cooled to 18deg4;C under alpha-stat. The dogs were assigned to 3 groups: antegrade CPB under alpha-stat (antegrade group) or RCP for 90 minutes under alpha-stat (RCP-alpha group) or pH-stat (RCP-pH group). In the RCP groups, the brain was perfused through the bilateral maxillary veins at 18°C, and perfusion flow was regulated at a sagittal sinus pressure of 20 to 25 mm Hg (mean, 22.5 ± 0.4 mm Hg). During perfusion, the proximal external cervical vein and the superior vena cava were clamped. The dog was sacrificed after 90 minutes of perfusion. In the antegrade group, systemic perfusion flow was regulated at 53 ± 5.7 mLákg–1ámin–1, leading to a perfusion pressure of 35 to 40 mm Hg during deep hypothermia. No intervention was used to control blood pressure during the study.
The following measurements were made. CBF of the cortex during perfusion was measured by the Doppler flowmeter. Although this method is useful for continuous monitoring, there appears to be no universal calibration factor and the measured value is affected by external illumination because of the small sample volume.4,5 Therefore, CBF was measured at one specific point during the procedure, and the probe was covered with black soft foam rubber to exclude external light.
Oxygen and CO2 tension (PO2 and PCO2) and pH in samples from the inflow to and outflow from the brain were analyzed at 37°C (ABL 4; Radiometer Medical, Br¿nsh¿j, Denmark) every 15 minutes. In this study, the inflow under RCP was the maxillary vein and the outflow the common carotid artery, while the reverse applied under antegrade CPB.
Plasma NOx levels were measured using ENO-200 (EiCOM, Kyoto, Japan). Nitrate was reduced to nitrite in a cadmium reduction column, and the nitrite was mixed with Griess reagent to form a purple azo dye in a reaction coil. The separation and reduction columns and the reaction coil were placed in a column oven set at 35°C. Color absorbance of the product dye at 540 nm was measured by a flowthrough spectrophotometer. Total nitric oxide metabolite levels were calculated as the sum of the nitrite and nitrate levels.3 Measurements were made in samples from the brain inflow and outflow every 15 minutes during deep hypothermic perfusion. To evaluate the effect of CPB itself and brain temperature, measurements were also made to inflow and outflow samples before CPB, under normothermic antegrade CPB, and under deep hypothermic antegrade CPB in all the dogs while under alpha-stat.
The values of blood gas analysis, CBF, and NOx were taken at RCP duration of 30 to 90 minutes to minimize the effects of perfusion flow manipulation and gas management. The values of CBF, cerebral metabolic rate for oxygen (CMRO2), and NOx were expressed as percentages of baseline values obtained before CPB. CMRO2 is calculated as oxygen (O2) extraction x CBF/100, where O2 extraction is the difference between inflow and outflow O2 content.
Systemic physiological data were initially analyzed using nonparametric multiple comparison (Kruskal-Wallis test); and if significance was proven, analysis of variance was done using multivariate comparison (Games-Howell test). The 2 RCP groups were compared using the Mann-Whitney U test. All data are presented as mean ± standard error of the mean. Differences at p < 0.05 are considered significant.
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RESULTS
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All blood pH, PO2, and PCO2 values are reported as measured at 37°C without correcting for body temperature (Tables 1 and 2
). Inflow PO2 did not differ between the 3 groups at any point during the study (Tables 1 and 2). Hemoglobin, PO2, O2 content, O2 extraction, and perfusion flow rate did not differ between the RCP groups (Table 2). CBF was significantly lower in the RCP-alpha group than in the RCP-pH and the antegrade groups, while CMRO2 was higher for RCP-pH than for RCP-alpha but showed no significant difference between either RCP group and the antegrade group (Figures 1A and 1B). CBF and O2 extraction were almost constant in each group during the study. Outflow NOx levels were significantly lower for RCP-pH than for RCP-alpha (Figure 1C). The ratio of outflow to inflow NOx levels tended to be higher for RCP-pH than for RCP-alpha, but not to a significant degree (Table 2). During cooling, outflow NOx levels and the ratio of outflow to inflow NOx levels before CPB, under normothermic CPB, and under deep hypothermic CPB were not significantly different (Table 3).
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Figure 1. (A) Cerebral blood flow of the cortex, (B) cerebral metabolic rate for oxygen, and (C) plasma total nitric oxide oxidation products in the brain outflow during retrograde cerebral perfusion (RCP) under alpha- or pH-stat or during antegrade perfusion. Data are expressed as percentages of baseline values obtained before cardiopulmonary bypass.
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Table 3. Plasma Levels of Total Nitric Oxide Oxidation Products During Cooling, as a Percentage of the Level Before Cardiopulmonary Bypass
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DISCUSSION
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RCP is useful because of easy deairing, its potential for limiting cerebral events and for flushing away deleterious neurological toxins, and the maintenance of aerobic metabolism.6 With regard to the clinical use of deep hypothermic CPB, the principal debate over PCO2 has centered on the respective advantages of alpha- and pH-stat management.
There are anatomical differences between humans and dogs. The canine maxillary vein drains blood from the whole head, snout, and cerebrum. There are competent venous valves in the external cervical vein at its proximal portion. The internal jugular vein is hardly developed. There are 2 cervical branches from the aortic arch: brachiocephalic artery and left subclavian artery. Bilateral common carotid arteries and the right subclavian artery arise from the brachiocephalic artery. Compared with humans, the internal carotid artery is very small in dogs. Therefore, in this study, the inflow to the brain under RCP was the maxillary vein and the outflow the common carotid artery, and we hypothesized that the brain circulation can be separated from the head circulation during RCP. In a preliminary study, blood gas analysis produced the same results between the internal and the common carotid artery as well as between the superior sagittal sinus and the maxillary vein.
We monitored the pressure in the superior sagittal sinus during RCP. The perfusion pressure was regulated at 20 to 25 mm Hg, which is considered optimal.7 RCP duration was set at 90 minutes because the safety limit of RCP was reported to be about this duration in humans.8
As it is necessary to control blood CO2 in the brain and heart under moderate hypothermia to minimize the effects of CO2, alpha-stat was used during cooling in all the groups. The systemic perfusion flow was kept around 50 mLákg–1ámin–1, resulting in a perfusion pressure of 35 to 40 mm Hg at 18°C. The perfusion rate used was determined based on reported optimal rates during deep hypothermia.9,10 The optimal rate needs to be studied further from various perspectives.
There have been several reports on cortical CBF under RCP.11,12 However, none have studied it in relation to CO2 management and in comparison with antegrade cerebral perfusion. Perfusion flow to the brain under RCP in humans has been reported to be significantly different between alpha- and pH-stat.13 In our study, perfusion flow to the brain did not differ between the RCP groups.
From our results, pH-stat appears to be neuroprotective during RCP in that cerebral oxygen supply was enhanced and its use suppressed. Alpha-stat is regarded as preferable to avoid the so-called "luxury perfusion" under pH-stat. However, under pH-stat, this is considered a necessary compensatory mechanism for the leftward shift of the oxyhemoglobin dissociation curve induced by hypothermia when perfusion flow is limited compared with antegrade perfusion.14 Our results indicate that CBF under pH-stat RCP is not excessive for global tissue oxygen demand compared with antegrade perfusion under alpha-stat. We used CMRO2 to evaluate global oxygen metabolism in the brain. It is dependent on CBF and O2 extraction, both of which were almost constant in each group during this study.
Attenuation of NOx levels in the brain may play a role in protecting against NO-induced neuronal damage under deep hypothermic RCP.1–3 Hypothermic neuroprotection during cerebral hypoxia might be attributable to amelioration of damage by a reduction in presynaptic excitatory amino acid release.15 CPB itself, independent of temperature, may have an effect on plasma NOx levels in the brain. It is unclear in this study whether cooling or CPB itself contributes more to plasma NOx in the brain.
In the brain, neuronal, inducible, and endothelial NO synthases (nNOS, iNOS, eNOS) are thought to perform overlapping roles. It is speculated that pH-stat decreases calcium influx by reducing NMDA-receptor activation, glutamate neurotoxicity, and nNOS-derived neuronal injury. It has been reported that iNOS activation causes elevation of plasma NOx in the brain16 and that endothelial function is attenuated during deep hypothermia.17 Deep hypothermic RCP under pH-stat may be neuroprotective by reducing NO metabolism. The relationship between pH management and iNOS/eNOS ratio under deep hypothermic RCP needs to be studied further.
In addition, extracellular acidosis may have a brain-protective role.18 Experimental studies suggest that under pH-stat, extracellular acidosis may inhibit cerebral excitotoxicity. Moreover, CO2 appears to exert a direct suppressive effect on CMRO2, independent of CBF.19 It is also possible that increased CBF under pH-stat results in the reduction of NOx in the outflow.
The sample size of 5 dogs in each group is small. However, the values of blood gas analysis, CBF, and NOx were taken at RCP duration of 30 to 90 minutes and were almost constant within each group during the study. Moreover, we compared CBF, CMRO2, and NOx data expressed as percentages of baseline values.
There are some difficulties in measuring brain metabolism, especially during RCP, in the dog. Besides anatomical differences between humans and dogs, there are venovenous shunts in the canine brain. One can argue that the dog is not necessarily a good model, but most mammalian species suffer from the same drawbacks as the dog.
The evaluation of the temporal effect of RCP duration on flow distribution in the brain in relation to CO2 management and histological study were not carried out in this study. They need to be studied as they may offer significant insights into brain protection.
In conclusion, CO2 management is crucial for brain protection under deep hypothermic RCP. This study shows that reduction of brain plasma NOx may decrease NO-induced neuronal damage. In addition, it is possible that increased CBF under pH-stat leads to reduced NOx production in the brain. In RCP, pH-stat is therefore better than alpha-stat in terms of CBF and NO metabolism in the brain.
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ABSTRACT
INTRODUCTION
