Ann Thorac Surg 1996;62:191-198
© 1996 The Society of Thoracic Surgeons
Original Articles: Cardiovascular
Moderate Hypothermia Reduces Cardiopulmonary Bypass-Induced Impairment of Cerebrovascular Responses to Platelet Products
Alon Stamler, MD,
Steven Y. Wang, MD, PhD,
Jianyi Li, MD,
Robert L. Thurer, MD,
Frederick J. Schoen, MD, PhD,
Frank W. Sellke, MD
Division of Cardiothoracic Surgery, Department of Surgery, aBeth Israel Hospital and Department of Pathology, bBrigham and Women's Hospital Boston, Massachusetts USA
Accepted for publication March 12, 1996.
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Abstract
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Background. The purpose of this study was to determine whether cerebral cortical microvascular responses to platelet-derived vasoactive substances are altered after normothermic cardiopulmonary bypass (CPB), and whether these alterations are modified by moderate hypothermia.
Methods. Pigs were placed on normothermic CPB (37°C) for 2 hours and then perfused off CPB with normothermic blood for either 15 minutes (n = 8) or 60 minutes (n = 6). Another group was placed on moderately hypothermic CPB (25°C) for 2 hours and then perfused off CPB at 37°C for 15 minutes (n = 6). Alpha-stat pH management was used. In vitro responses of isolated cortical cerebral arterioles (90 to 170 µm internal diameter) to platelet-derived vasoactive substances were examined in a pressurized no-flow state with video-microscopy. Microvessels from noninstrumented pigs (n = 14) were used as controls for in vitro studies.
Results. Cerebrovascular resistance and internal carotid artery blood flow were similar 15 minutes after CPB in both normothermic and hypothermic groups. However, relaxations of microvessels to adenosine 5' diphosphate or serotonin were reduced in vessels from both groups. One hour of after CPB cerebral perfusion did not change this pattern of altered vascular reactivity. Hypothermia caused a partial but significant reduction in impairment of responses to adenosine 5' diphosphate and serotonin. Microvascular relaxation to the endothelium-independent agent sodium nitroprusside and contraction to a thromboxane A2 analog were similar in all experimental groups, suggesting normal vascular smooth muscle responsiveness.
Conclusions. This study demonstrates that normothermic extracorporeal circulation reduces endothelium-dependent relaxation responses to products of platelet activation in the cerebral microcirculation. Moderate hypothermia attenuates the CPB-induced impairment of endothelium-dependent relaxation, but has no effect on baseline cerebral blood flow after rewarming.
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Introduction
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Neuropsychologic dysfunction can be a cause of significant mortality and morbidity after cardiac operations. Whereas overt stroke occurs in less than 5% of cardiac surgical patients [1], subtle postoperative neurologic and cognitive dysfunction is detectable after up to 79% of cardiac operations [2–5]. The cause of this neuropsychologic dysfunction is unknown but it has been attributed in large part to the interruption of cerebral perfusion by either arterial hypotension, altered autoregulation, nonpulsatile cerebral perfusion, or gaseous and particulate microembolization during cardiopulmonary bypass (CPB) [6].
Recently, it has become better appreciated that the adverse effects of CPB may be due to a generalized intravascular inflammatory response to extracorporeal circulation [7], which leads to increased vascular permeability [8] and in many cases to both vascular and parenchymal dysfunction [9]. Complement and other humoral factors are activated during CPB [7, 10–12], which may impact on normal physiologic parameters. In addition, both leukocytes and platelets are activated during extracorporeal circulation [11]. When these cellular blood components are activated, they release vasoactive substances that may dramatically alter vasomotor tone. Activation of platelets causes production and release of many substances including thromboxane A2, adenosine 5' diphosphate (ADP), and serotonin (5-HT) from dense storage granules [11, 13]. Activation of leukocytes may result in the release of kinins, leukotrienes, and histamine in addition to causing direct vascular injury [14]. Therefore, platelet activation may alter vasomotor tone and affect perfusion to the brain and other end organs. Whereas most studies examining the effects of products of platelet and leukocyte activation have been performed on the coronary circulation, there is no reason that the cerebral circulation is not affected.
Regulation of cerebral blood flow is complex and is influenced by autoregulatory, metabolic, and neurohumoral factors. The endothelium is known to regulate vascular tone in the cerebral and other vascular beds [15]. Kimura and co-workers [16] recently showed that inhibition of endothelium-derived nitric oxide produces substantial contraction of cerebral vessels, suggesting a role of nitric oxide in maintaining cerebral vascular tone. Previously we have found that autonomic neuronal regulation of cerebral perfusion is altered after normothermic CPB [17]. The effect of normothermic and hypothermic CPB on endothelium-dependent relaxation to humoral platelet-derived substances has not been examined. Our general hypothesis is that cerebral blood flow regulation is altered after CPB, and that these changes may impact on neuropsychologic recovery after cardiac operation. Because aggregating platelets may cause very high local concentrations of ADP, thromboxane, and 5-HT, especially in areas of atherosclerotic plaque formation, it is possible that increased concentrations of both circulating and locally released platelet-derived substances will alter cerebral vascular tone. Furthermore, if endothelium-dependent relaxation is altered after CPB, it is plausible that permeability and other indices of endothelial function are also impaired.
The aim of this study was to investigate whether cerebral blood flow and cortical endothelial cell function are altered as a consequence of normothermic CPB and to study specifically the effects of complement activation on the reactivity of cerebral arterioles. In addition, the modulating influence of moderate hypothermia on vasomotor alterations was examined.
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Material and Methods
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Animal Preparation
Yorkshire pigs (20 to 25 kg) of either sex were premedicated intramuscularly with ketamine (10 mg/kg) and anesthetized intravenously with alpha-chloralose and urethane (60 mg/kg and 300 mg/kg initially and 15 mg/kg and 75 mg/kg every 60 minutes as needed, respectively). Pigs were intubated tracheally and mechanically ventilated. In the control group (n = 14), pigs were heparinized (500 U/kg). The animals were turned prone, and a 3- by 3-cm2 hole was made through the parietofrontal bone. Excessive bleeding was controlled using bone wax, and the overlying dura was excised. Tissue specimens of the cortical gray matter in the distribution of the middle cerebral artery were obtained and immediately placed in cold (4°C) MOPS buffer solution of the following composition (in mmol/L) : 145.0 NaCl, 4.7 KCL, 2.0 CaCL, 1.2 MgSO4, 5.0 glucose, 2.0 pyruvate, 0.02 EDTA, 1.2 NaH2PO4, and 3.03-(N-morpholino) propanesulfonic acid (MOPS).
Animals were cared for in accordance with the guidelines established by the Beth Israel Hospital Animal Care and Use Committee and the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985).
Cardiopulmonary Bypass Group
In 8 pigs, after induction of anesthesia and tracheal intubation, a longitudinal cervical dissection was performed exposing the right common carotid artery, the external carotid artery, internal carotid artery (ICA), and internal jugular vein. A vessel loop was placed around the external carotid artery. A 5F catheter was inserted into the common carotid and advanced to the proximal ICA. Another 5F catheter was inserted retrograde into the internal jugular vein and advanced into the venous sinus. Catheters were connected to fluid pressure lines attached to pressure transducers. An ultrasonic flowmeter (Transonic Systems Inc, Ithaca, NY) was placed around the proximal ICA. An 8F (Millar, Houston, TX) catheter was introduced from the femoral artery to the thoracic aorta for pressure measurement. The femoral vein was cannulated for vascular access. The chest was open through a sternotomy, and pigs were heparinized (500 U/kg and 250 U/kg after 90 minutes) and cannulated through the distal ascending aorta and the right atrium. Activated clotting time was measured and maintained more than 500 seconds. Total CPB was instituted using a bubble oxygenator (Bentley Bio-2, Baxter Healthcare, Irvine, CA) and a roller pump. An arterial filter (Bentley AF-1025, Baxter Healthcare) was inserted into the circuit distal to the roller pump. During CPB, pump blood flow was maintained between 80 and 90 mL • min-1 • kg-1 to maintain a mean systemic blood pressure more than 50 mm Hg. Nasopharyngeal and rectal temperatures were measured continuously and remained between 36 and 38°C in all pigs. Arterial blood gases (pH Blood gas analyzer 1306; Instrumentation Lab, Lexington, MA) were measured before initiation of CPB and at 15-minute intervals. Before and after CPB, adjustments were made in the ventilatory rate, tidal volume, and fractional concentration of oxygen to maintain the partial pressure of oxygen more than 100 mm Hg, pH between 7.35 and 7.45, and the partial pressure of carbon dioxide more than 35 and less than 45 mm Hg. During CPB, mean arterial pressure (MAP), mean jugular venous pressure (MJP), and unilateral ICA blood flow were monitored and recorded every 15 minutes. Cerebral vascular resistance was calculated as (MAP - MJP in mm Hg) / unilateral ICA blood flow (mL /min) x 79.9 and expressed as dynes • s-1 • cm-5. After 120 minutes of extracorporeal circulation, pigs were separated from CPB and the brains were perfused off CPB for 15 minutes with normothermic blood. A 3- by 3-cm2 burr hole was made through the parietofrontal bone. The overlying dura was excised. Specimens of cortical brain tissue were taken as described previously and immediately placed in cold MOPS buffer solution.
Cardiopulmonary Bypass and 1 Hour of Normal Cerebral Perfusion
In 6 pigs, a similar procedure was performed as described above. In this group, the brain was perfused with normothermic blood for 60 minutes after termination of CPB while maintaining systemic blood pressure above 50 mm Hg with fluid administration (Ringer's lactate). The brain tissue was then harvested as described above.
Hypothermic Cardiopulmonary Bypass
In 6 pigs, a similar procedure was followed, except after initiation of CPB, the temperature was decreased to 25°C. The period of cooling lasted approximately 20 minutes. This temperature was maintained until 30 minutes before the time of anticipated termination of CPB. At that time, rewarming to 37°C was initiated. During hypothermic CPB, alpha-stat for pH management was used. Pigs were separated from CPB and brains were perfused with normothermic blood for 15 minutes while maintaining systemic blood pressure above 50 mm Hg with fluid administration (Ringer's lactate). The brain tissue was then harvested as described above.
In Vitro Cerebral Microvessel Studies
Isolated cerebral arterioles (internal diameter, 90 to 170 µm) were dissected from the cortical gray matter using a 10 to 60x dissecting microscope (Olympus Optical, Tokyo, Japan). Microvessels were placed in a plexiglass isolated vessel chamber, cannulated with dual glass micropipettes measuring 30 to 80 µm in diameter and secured with 10-0 nylon monofilament suture (Ethicon, Somerville, NJ). MOPS buffer solution (pH 7.4) equilibrated with room air was continuously circulated through the vessel chamber and a reservoir containing 100 mL. The solution was maintained at 37°C by an external glass heat exchanger. Vessels were pressurized to 40 mm Hg in a no-flow state using a burette manometer filled with MOPS buffer solution. With an inverted microscope (40-200x IMT-2, Olympus Optical) connected to a video camera, the vessel image was projected onto a black and white television monitor. An electronic dimension analyzer (Living System Instrumentation, Burlington, VT) was used to measure internal lumen diameter. Measurements were recorded with a stripchart recorder (Western Graphtec, Irvine, CA). Vessels were allowed to equilibrate for 30 minutes in MOPS buffer solution before a pharmacologic intervention.
In Vitro Study Protocol
After equilibration of the microvessels for at least 30 minutes, vessels were precontracted by 30% to 40% of the baseline diameter with the thromboxane A2 analog U46619 (0.1 to 1.0 µmol/L). U46619 was chosen as the precontraction agent because it produces consistent sustained contraction of porcine microvessels [18–20]. After stabilization of the precontracted diameter, 5-HT (1 nmol/L to 0.1 mmol/L), ADP (1 nmol/L to 0.1 mmol/L), or sodium nitroprusside (SNP, 1 nmol/L to 0.1 mmol/L) were applied extraluminally. Adenosine 5' diphosphate is an endothelium-dependent vasodilator, whereas 5-HT has an endothelium-dependent relaxing effect and a direct contractile effect on vascular smooth muscle. Sodium nitroprusside is an endothelium-independent cyclic guanosine monophosphate-mediated vasodilator. The order of administration was random. The vessels were washed three times with MOPS buffer solution and allowed to equilibrate for 10 to 15 minutes between drug interventions. Two or three microvessels were examined from each animal, and one to three interventions were performed on each vessel.
In selected experiments, the cyclooxygenase inhibitor indomethacin (10 µmol/L, n = 6) or the nitric oxide synthase inhibitor NG-nitro-L-arginine (30 µmol/L, n = 6) was added to the vessel chamber reservoir. In addition, the effects of alternate pathway complement activation on the cerebral microvascular responses to ADP, 5-HT, and SNP were examined. In this group, 10% porcine serum and zymosan (1 mg/10 mL) was applied intraluminally for 30 minutes before the application of vasoactive agents.
Morphologic Analysis: Transmission Electron Microscopy
Selected vessel specimens were examined with transmission electron microscopy to determine whether the integrity of the vascular smooth muscle and endothelium was preserved after 120 minutes of normothermic CPB. In separate experiments (n = 2), after 2 hours of CPB and 15 minutes of cerebral perfusion off CPB, 500 mL of Karnovsky's solution (cacodylate buffer, 0.1 mol/L, 2.5% glutaraldehyde, and 2% paraformaldehyde, pH 7.2.) at room temperature was infused into the ICA through a 14-gauge catheter at a pressure of 90 mm Hg. Perfusion was continued for 5 minutes. The brain was then harvested, immersed in additional Karnovsky's solution, and refrigerated. Representative specimens of microvessels were postfixed in osmium tetroxide, dehydrated, and embedded in polybed 812 (Polyscience Inc, Warrington, PA). Ultrathin sections stained with uranyl acetate were examined in a model JEM 100CX transmission electron microscope (JEOL, Cranford, NJ).
Drugs
The 5-HT, ADP, U46619, NG-nitro-L-arginine, SNP, indomethacin, and zymosan were obtained from Sigma Chemical (St. Louis, MO). The 5-HT, ADP, NG-nitro-L-arginine, and SNP were dissolved in ultrapure distilled water. U46619 was dissolved in ethanol to make a 10 mmol/L stock solution. Indomethacin was dissolved in minimal ethanol aqueous solution to make a 10 mmol/L solution. All stock solutions were stored at -20°C. All dilutions were prepared daily.
Data Analysis
The response of microvessels to each agent was examined only once in each animal. Therefore, each animal served as one sample. The data were pooled from each dose response in each experimental group and an average was calculated. The relaxation responses were expressed as percentage relaxation of the U46619-induced precontraction of the vessel diameter. Values were expressed as mean ± standard error of mean. Values of MAP, ICA blood flow, and cerebral vascular resistance between groups were compared at each time point using Student's t test. Comparisons of in vitro dose responses of all experimental groups were performed by two-factor (drug concentration, experimental group) ANOVA for repeated measures. Whenever significance was indicated, Fisher's multiple comparison test was used post-hoc to make comparisons between specific groups. A p value of less than 0.05 was considered to be statistically significant.
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Results
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Hemodynamic Characteristics
The MAP, cerebral vascular resistance, and unilateral ICA blood flow are shown in Figure 1
. The initiation of CPB coincided with a decrease in MAP in both normothermic and hypothermic CPB groups. The MAP remained relatively constant during and after termination of CPB in the normothermic pigs. However, pigs in the hypothermic CPB group developed a progressive increase in MAP during the rewarming period. The cerebral vascular resistance decreased immediately upon onset of bypass and decreased slightly during normothermic CPB. Hypothermic CPB was associated with an attenuated decrease in baseline cerebral vascular resistance, but the slow progressive decrease was still observed. The ICA artery blood flow decreased slightly with the onset of normothermic CPB and then returned to baseline value and remained relatively constant. Hypothermic CPB was associated with reduced cerebral blood flow during the hypothermic period. After completion of rewarming and 15 minutes after separation from CPB, cerebral vascular resistance, and ICA blood flow were similar in the normothermic and hypothermic CPB groups.
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Fig 1. . (A) Plot of mean arterial blood pressure (MAP); (B) percentage change in cerebrovascular resistance (CVR); and (C) percentage change in internal carotid artery (ICA) blood flow in the normothermic cardiopulmonary bypass (CPB) and hypothermic CPB groups as a function of time. T = -120 minutes and "On CPB" are time of onset of CPB. T = 0 and "Off CPB" are time of termination of CPB. Values are mean ± standard error of the mean. (*p < 0.05 between groups.)
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Vessel Characteristic
Cerebral microvessels averaged 143 ± 11 µm in the control group, 146 ± 7 µm in the normothermic CPB 15-minute group, 137 ± 6 µm in the normothermic CPB 60-minute group, and 134 ± 6 µm in the hypothermic CPB 15-minute group. Percent contraction after application of thromboxane A2 analog U46619 was 34 ± 1 in control vessels, 35 ± 1 in vessels in the normothermic CPB 15-minute group, 33 ± 1 in the normothermic CPB 60-minute group, and 34 ± 3 in the hypothermic CPB 15-minute group. The concentration of U46619 required to induce this degree of contraction was similar in all groups (log molar[U46619] = -6.1 ± 0.1 (control), -6.1 ± 0.4 (normothermic CPB 15-minute), -6.2 ± 0.1 (normothermic CPB 60-minute), and -6.0 ± 0.1 (hypothermic CPB 15-minute).
In Vitro Response to Adenosine 5' Diphosphate
Adenosine 5' diphosphate elicited a significant relaxation response in control vessels. After 2 hours of normothermic CPB and 15 minutes of normal cerebral perfusion off CPB, the relaxation response was significantly reduced. The relaxation response was further reduced after 60 minutes of perfusion off CPB. Hypothermia partially preserved the endothelium-dependent relaxation to ADP (p < 0.05 versus normothermic CPB 15-minute group only at 100 µmol/L; Fig 2). In the presence of the cyclooxygenase inhibitor indomethacin, the ADP-induced relaxation was reduced a small but significant amount compared to the relaxation response in nontreated control vessels. In the presence of nitric oxide synthase inhibitor NG-nitro-L-arginine, ADP-induced relaxation was markedly reduced (Fig 3).
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Fig 2. . Plot of in vitro responses to adenosine 5' diphosphate (ADP). Microvessels (90 to 170 µm) were harvested from noninstrumented pigs (control, n = 8), after 2 hours of normothermic (37°C) cardiopulmonary bypass (CPB) followed by either 15 minutes (normothermic CPB 15-minute, n = 8) or 60 minutes (normothermic CPB 60-minute, n = 6) of normothermic perfusion after termination of CPB, or after 2 hours of moderately hypothermic (25°C) CPB followed by 15 minutes of normothermic perfusion off CPB (hypothermic CPB 15-minute, n = 6). Responses are percentage relaxation of the U46619-induced contraction. Values are mean ± standard error of the mean. (*p < 0.05 versus control.  p < 0.05 versus normothermic CPB 60-minute group.)
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Fig 3. . Plot of in vitro response to adenosine 5' diphosphate (ADP) in the presence of the nitric oxide synthase inhibitor NGnitro-L-arginine (Nitro-L-Arginine) (30 µmol/L, n = 6) or the cyclooxygenase inhibitor indomethacin (10 µmol/L, n = 8) for 20 minutes. Control vessels (n = 6) were untreated. Responses are percentage relaxation of the U46619-induced contraction. Values are mean ± standard error of the mean. (*p < 0.05 versus respective control.)
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In Vitro Response to Serotonin
The 5-HT caused a significant relaxation of control cerebral microvessels. After 2 hours of normothermic CPB and either 15 or 60 minutes of additional perfusion after separation from CPB, this relaxation response was significantly reduced. Hypothermia completely preserved the relaxation response to 5-HT (Fig 4). In the presence of cyclooxygenase inhibitor indomethacin, 5-HT-induced relaxation was not altered. In the presence of NG-nitro-L-arginine, serotonin caused a significant contractile response in control microvessels (Fig 5).
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Fig 4. . Plot of in vitro responses to serotonin (5-HT). Microvessels (90 to 170 µm) were harvested from noninstrumented pigs (control, n = 8), after 2 hours of normothermic (37°C) cardiopulmonary bypass (CPB) followed by either 15 minutes (normothermic CPB 15-minute, n = 8) or 60 minutes (normothermic CPB 60-minute, n = 6) of normothermic perfusion after termination of CPB, or after 2 hours of moderately hypothermic (25°C) CPB followed by 15 minutes of normothermic perfusion off CPB (hypothermic CPB 15-minute, n = 6). Responses are percentage relaxation of the U46619-induced contraction. Values are mean ± standard error of the mean. (*p < 0.05 versus control, p < 0.05 versus normothermic CPB 60-minute group.)
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Fig 5. . Plot of in vitro response to serotonin (5-HT) in the presence of the nitric oxide synthase inhibitor NGnitro-L-arginine (Nitro-L-Arginine) (100 µmol/L, n = 6) or the cyclooxygenase inhibitor indomethacin (10 µmol/L, n = 7) for 20 minutes. Control vessels (n = 6) were untreated. Responses are percentage relaxation of the U46619-induced contraction. Values are mean ± standard error of the mean. (*p < 0.05 versus respective control.)
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Endothelium-independent Responses to Nitroprusside and Thromboxane
Relaxation of cerebral microvessels to the endothelium-independent vasodilator SNP was similar in all experimental groups, indicating no alteration in the ability of cerebral vascular smooth muscle to relax after 2 hours of normothermic CPB and after CPB brain perfusion (Fig 6). The thromboxane A2 analog U46619 (1.0 µmol/L) induced a contraction response of 34 ± 1% in control vessels, 35 ± 1% in vessels from the normothermic CPB 15-minute group, 33 ± 1% in the normothermic CPB 60-minute group, and 34 ± 3% in the hypothermic CPB 15-minute group.
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Fig 6. . Plot of in vitro responses to sodium nitroprusside. Microvessels (90 to 170 µm) were harvested from noninstrumented pigs (control, n = 6), after 2 hours of normothermic (37°C) cardiopulmonary bypass (CPB) followed by either 15 minutes (normothermic CPB 15-minute, n = 7) or 60 minutes (normothermic CPB 60-minute, n = 6) of normothermic perfusion after termination of CPB, or after 2 hours of moderately hypothermic (25°C) CPB followed by 15 minutes of normothermic perfusion off CPB (hypothermic CPB 15-minute, n = 6). Responses are percentage relaxation of the U46619-induced contraction. Values are mean ± standard error of the mean.
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Effect of Alternate Pathway Complement Activation
Exposure of microvessels to zymosan-induced complement-activated porcine serum (10%) significantly reduced the relaxations to ADP and 5-HT in control microvessels (Fig 7). The response to SNP was not altered after exposure of microvessels to complement activated serum (76 ± 4% and 74 ± 5% relaxation at 10 µmol/L SNP in control and complement-exposed vessels, respectively).
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Fig 7. . Plot of in vitro responses to adenosine 5'diphosphate (ADP) and serotonin (5-HT), after intraluminal application of zymosan-induced complement activated serum (complement, n = 6) for 30 minutes or in its absence (control, n = 6). Responses are percentage relaxation of the U46619-induced contraction. Values are mean ± standard error of the mean. (*p < 0.05 versus respective control.)
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Transmission Electron Microscopy
Transmission electron microscopy of representative microvessels from two brains after 2 hours of normothermic CPB and 15 minutes of perfusion off CPB showed normal vascular morphology without endothelial detachment, necrosis, adherent inflammatory cells, or otherwise morphologically apparent injury (Fig 8).
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Fig 8. . Ultrastructure of representative perfusion-fixed small arterial cortical cerebral vessel from a pig placed on normothermic cardiopulmonary bypass (37°C) for 2 hours and perfused with normothermic blood off cardiopulmonary bypass for 15 minutes. The arteriole illustrated has intact endothelial cells with near-normal morphology and without loss of attachment to the underlying basement membrane, necrosis, or other evidence of injury. No inflammatory cells are adherent. (x7,200 before 50% reduction; bar = 2 µm).
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Comment
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The principal finding of this investigation is that normothermic CPB markedly alters cortical cerebral microvascular responses to products of platelet activation. The impairment in vascular reactivity was selective in that only responses to endothelium-dependent vasodilators (ADP and 5-HT) were significantly altered, whereas responses to agents that act directly through the vascular smooth muscle (SNP and thromboxane A2) did not change. Importantly, the reduced endothelium-dependent relaxation was further impaired or at least not improved after prolonged (1 hour) cerebral perfusion after separation of pigs from extracorporeal circulation. This suggests that the effects of CPB on vascular reactivity are not reversed soon after termination of CPB. Moderately hypothermic CPB partially but significantly preserved endothelium-dependent relaxation to ADP and 5-HT.
Platelets are activated immediately after the start of CPB. This activation is triggered by direct surface contact of blood with the oxygenator and extracorporeal circuit, abnormal sheer stress, mechanical lysis, and chemical stimuli. The altered endothelium-dependent relaxations to ADP and 5-HT are not specific to extracorporeal circulation. After exposure of vessels to cardioplegia or endotoxin, in addition to extracorporeal circulation, endothelium-dependent relaxation may be impaired or converted to a contractile response [18–21]. The contractile response observed to 5-HT is likely in part attributable to the increased release of a constrictor prostaglandin substance, as the contractile vascular responses after either cardioplegia or endotoxemia are attenuated after inhibition of cyclooxygenase [18, 20, 21].
Relaxations of cerebral microvessels to ADP and serotonin are markedly reduced after treatment with NG, nitro-L-arginine, whereas relaxation of the vessels to SNP are not affected. Cyclooxygenase inhibition has a small effect on ADP-induced relaxation, but no effect on that elicited by 5-HT. This suggests that in the porcine cerebral microvasculature, relaxations to ADP and 5-HT are predominately mediated by endothelium-dependent mechanisms and largely through the release of nitric oxide with a secondary contribution of prostaglandin release and perhaps other mechanisms such as hyperpolarization.
Activation of complement likely plays a significant role in mediating endothelial dysfunction during CPB, as vessels exposed to complement-activated serum had significantly reduced relaxation responses to ADP and 5-HT, but had no reduction in the endothelium-independent relaxation to SNP. The alternate complement pathway is activated during exposure of blood to foreign surfaces such as the extracorporeal circuit and oxygenator [10] or when porcine serum comes in contact with zymosan [22]. Complement activation during CPB leads to formation of anaphylatoxin C3a and C5a mainly through the alternative pathway. CPB-induced inflammatory responses involves adhesion of complement-activated neutrophils to endothelial cell and subsequent release of neutrophil-derived cytotoxic mediators that may contribute to end organ dysfunction [12, 23]. In an experimental model of normothermic extracorporeal circulation, a clear linear increase in both C3a and C5b-9 has been demonstrated [7]. Moore and colleagues [24] examined the effect of hypothermia on complement activation during CPB and found that it decreased levels of activated complement fragments. Because activated complement was found to decrease endothelium-dependent relaxation, it is plausible that the beneficial effect of hypothermia is due to the attenuating effect of hypothermia on complement activation. Despite the above findings, it is likely that other factors such as oxygen-derived free radicals, cytokines, activated leukocytes, receptor desensitization, and the release of cytotoxic and proteolytic enzymes are also involved in cerebral vascular dysfunction after CPB.
Cerebral vascular endothelial nitric oxide has been investigated in recent years, yet there is no clear understanding of its function after insults such as ischemia or CPB. Endothelium-derived nitric oxide is important in the regulation of basal cerebral vasomotor tone [13] and mediates the response of cerebral blood vessels to many stimuli [15, 25]. Tanaka and co-workers [26] demonstrated that the systemic administration of a nitric oxide synthase inhibitor reduced cerebral blood flow in rats. Iwamoto and colleagues [27] found that inhibition of nitric oxide synthase reduces the resting cerebral blood flow in sheep. These observations suggest that nitric oxide participates in the maintenance of basal tone of cerebral circulation [25]. In addition to a direct vasomotor action, nitric oxide can modulate cerebral blood flow indirectly by inhibiting vascular injury due to leukocyte activation or by decreasing platelet activation [25, 28].
In summary, the present study demonstrates that CPB and cerebral perfusion after CPB alter the reactivity of the cerebral microvasculature by reducing endothelium-dependent relaxation mechanisms to products of platelet activation. Hypothermia partially prevented these alterations in endothelium-dependent relaxation. The role of alternate pathway complement activation in causing changes in vascular reactivity is suggested but not proven by the present study. Future studies will directly examine if activation of the alternate complement pathway during CPB is a cause of the endothelial dysfunction during extracorporeal circulation with the use of antibodies to C5a and C8, or other less specific inhibitors of the complement cascade. In addition, other potential pathologic processes contributing to vascular dysfunction after CPB such as the generation of oxygen-derived free radicals and leukocyte activation will be examined. It is important to note that baseline cerebral perfusion was not altered by normothermic or hypothermic CPB. Thus, it is not known for certain if the impaired endothelial function contributes to changes in blood flow to the brain after cardiac operation, or if these altered responses to products of platelet activation are of any clinical relevance. However, as the endothelium is known to modulate vascular tone, it is probable that the altered simulated responses may contribute, at least in part, to changes in the control of cerebral perfusion after cardiac operation.
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Acknowledgments
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This study was supported by National Heart, Lung, and Blood Institute grant HL 46716 and American Heart Association, Massachusetts Affiliate grant 13-501-912.
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Footnotes
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Address reprint requests to Dr Sellke, Division of Cardiothoracic Surgery, Beth Israel Hospital, Dana 905, 330 Brookline Ave, Boston, MA 02215.
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Abstract
Introduction
