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J Thorac Cardiovasc Surg 1994;108:1092-1099
© 1994 Mosby, Inc.
CARDIOPULMONARY BYPASS, |
Boston, Mass.
Supported by National Heart, Lung, and Blood Institute grant HL 46716, American Heart Association (Massachusetts Affiliate) grant 13-501-912, and a grant from the American College of Chest Physicians.
Received for publication Feb. 25, 1994. Accepted for publication June 14, 1994. Address for reprints: Frank W. Sellke, MD, Division of Cardiothoracic Surgery, Beth Israel Hospital, Dana 905, 330 Brookline Ave., Boston, MA 02215
Abstract
Total cardiopulmonary bypass with associated reduced pulmonary blood flow causes significant alterations of endothelium-dependent pulmonary microvascular responses after resumption of normal perfusion. To determine if this change in pulmonary vascular reactivity may influence the responses of pulmonary arterioles to protamine and histamine, we examined isolated pulmonary microvessels after cardiopulmonary bypass. Sheep were heparinized, cannulated, and placed on either total bypass without ventilation or partial bypass (70% of baseline pulmonary arterial flow) with continued ventilation. After 90 minutes, sheep were separated from cardiopulmonary bypass and the lungs were perfused normally for 60 minutes. Vessels from noninstrumented sheep were used as controls. Peripheral pulmonary arterioles (90 to 190µm) were cannulated, pressurized (20 mm Hg) in a no-flow state, and examined with video microscopy. After precontraction of vessels with the thromboxane A2analog U46619 by 18% to 25% of the baseline diameter, vasoactive agents were applied. Protamine sulfate, histamine, heparin, and the protamine-heparin complex caused significant dose-dependent relaxations of control pulmonary microvessels. These relaxation responses were substantially reduced or converted to contractile responses in endothelium-denuded vessels, which suggests that these relaxations are mediated through endothelium-dependent mechanisms. After partial bypass, responses to protamine and histamine were slightly reduced compared with the respective responses of control vessels, whereas the relaxation to protamine-heparin complex was not significantly altered. After total bypass, relaxation responses to protamine and protamine-heparin complex were markedly reduced, whereas histamine induced contraction of pulmonary microvessels. Endothelium-independent relaxation to sodium nitroprusside was not affected by partial cardiopulmonary bypass and was slightly reduced after total bypass. A reduced direct vascular relaxation response to protamine and increased contractile response to histamine (or other humoral substances released during the systemic administration of protamine sulfate) may contribute to the elevation of pulmonary vascular resistance during infusion of protamine after cardiopulmonary bypass. (J THORACCARDIOVASCSURG1994;108:1092-9)
Previous studies have shown that total cardiopulmonary bypass (CPB) with associated reduced pulmonary perfusion causes significant alterations of endothelium-dependent pulmonary microvascular responses because of the increased release of constrictor prostanoid substances and the reduced release of endothelium-derived nitric oxide. 
1 The genesis of this phenomenon is not known but maybe due to tissue ischemia, hypoxia, 2 or systemic inflammation associated with decreased pulmonary arterial flow and cessation of ventilation during CPB.
Protamine sulfate can cause detrimental hemodynamic effects including systemic hypotension, 3,4 cardiogenic shock, and pulmonary hypertension. 5,6 The mechanism causing these effects has not been determined, but several possibilities exist: (1) Protamine sulfate or protamine-heparin complexes have direct vasoactive properties on the smooth muscle of coronary and pulmonary vessels; (2) protamine sulfate may cause indirect vasoactivity because of endothelium-dependent properties in vessels in the two vascular beds; or (3) protamine administration during reversal of heparin anticoagulation may cause the synthesis or release of vasoactive substances from leukocytes, vascular tissue, or other sites. Two thirds of the protamine composition is L-arginine, a precursor of nitric oxide, 7 which is the biologic active component of endo thelium-derived nitric oxide. 8 Systemic infusion of L-arginine has been reported to cause hypotension. 9 Protamine can cause the release of endothelium-derived nitric oxide from systemic arteries, 10 possibly as a result of the large amount of L-arginine in protamine that can act as a substrate and be converted into nitric oxide in the endothelial cells, or more likely indirectly through activation of nitric oxide synthase. Finally, protamine or the pro tamine-heparin complex may act on blood elements such as leukocytes and cause the release of vasoactive substances such as prostaglandins, leukotrienes, and histamine. 3
The purpose of the present study was to examine the effects of total and partial CPB with associated reduced pulmonary perfusion on pulmonary microvascular responses to protamine and histamine. Responses of isolated pulmonary arterial microvessels (<190 µm) were examined, because these vascular segments provide the majority of regulation of pulmonary perfusion. 11
METHODS
Experimental preparation
Dorset-Rambouillet cross-bred sheep of female sex weighing 25 to 35 kg (mean 29 ± 0.6 kg) were anesthetized with intravenous 
-chloralose (80 mg/kg) and urethane (500 mg/kg). The animals were grouped into three categories.
In the total CPB group (n = 7), animals were tracheally intubated and their lungs mechanically ventilated (Harvard Apparatus, Millis, Mass.). Arterial blood gas and pH measurements were performed at regular intervals during the experiment and were maintained within physiologic limits (pH 7.35 to 7.42, oxygen tension > 100 mm Hg, < 300 mm Hg, carbon dioxide tension < 45 mm Hg, > 35) by adjusting the inspired oxygen tension, ventilation rate, and tidal volume. Systemic blood pressures were monitored by percutaneous cannulation of the femoral artery. A midline sternotomy was performed, and after systemic heparinization (400 Iu/kg), cannulas were placed in the right atrium and aorta. The extracorporeal circuit consisted of a standard roller pump (Cardiovascular Instrument Corp., Wakefield, Mass.) and bubble oxygenator (Bentley Bio-2, Baxter Healthcare Corp., Irvine, Calif.). An arterial filter (Bentley AF-1025, Baxter) was inserted into the circuit distal to the roller pump. Pulmonary artery and left atrial pressures were monitored with cannulas connected to a transducer. Pulmonary artery blood flow was measured with a Doppler flow probe (Transonic Systems, Inc., Ithaca, N.Y.). The circuit was primed with Ringer's lactate solution (25 ml/kg). Animals were placed on total CPB. After initial stabilization, the pulmonary artery was clamped to assure total absence of blood flow through the pulmonary artery, and ventilation was discontinued. Pump flow was adjusted from 80 to 100 ml/kg per minute to maintain aortic pressure between 50 and 75 mm Hg. After a period of 90 minutes of total CPB, ventilation was reestablished and the pulmonary artery clamp was removed. The lungs were reperfused for 60 minutes and normal pulmonary circulation was reestablished by increasing cardiac filling. Sheep were separated from CPB in all experiments. Inotropic and vasoactive agents were not used in any animals to support the circulation. Segments of the lung were excised from multiple peripheral sites and microvessels were obtained for in vitro studies.
In the partial CPB group (n = 8) after induction of anesthesia, the same procedure was followed as in the total CPB group, except only one third of estimated cardiac output was allowed to flow through the extracorporeal circuit. This limitation was accomplished by measuring the pulmonary artery flow with a Doppler flow probe before CPB and reducing it by one third by adjusting the venous drainage after establishing partial CPB. The lungs were ventilated during partial CPB. After 90 minutes, CPB was terminated and the lungs were perfused normally for 60 minutes. Peripheral segments of the lung were excised and microvessels were obtained for in vitro studies.
In the control group (n = 18), after induction of anesthesia, animals were heparinized and a sternotomy was performed. Peripheral segments of the lung tissue were excised and microvessels were obtained for in vitro studies. All animals were killed by exsanguination.
The excised lung tissue was immediately placed in cold (approximately 4° C) Krebs buffer solution of the following composition: NaC1, 118.3 mmol/L, KC1 4.7 mmol/L, CaC12 2.5 mmol/L, MgSO4 1.2 mmol/L, KH2PO4 1.2 mmol/L, NaHCO3 and glucose 11.1 mmol/L.
Animals were cared for in accordance with the guidelines established by the Beth Israel Hospital's Animal Care and Use Committee and those prepared by the Committee on the Care and Use of Laboratory Animals of the Institute of Animal Resources, National Research Council (NIH publication No. 86-23, revised 1985).
In vitro pulmonary microvascular studies
Microarterial vessels were carefully dissected from lobular branches of the pulmonary artery with a 10-60x dissecting microscope (Olympus Optical, Tokyo, Japan). These vascular segments varied in size from 90 to 190 µm in diameter and from 1 to 2 mm in length. Vessels were placed in an isolated Plexiglas organ chamber (University of Iowa Medical Instrumentation, Iowa City, Iowa), cannulated with dual glass micropipettes measuring 30 to 80 µm in diameter, and secured with 10-0 nylon monofilament suture (Ethicon, Inc., Somerville, N.J.). Oxygenated (95% oxygen, 5% carbon dioxide) Krebs buffer solution warmed to 37° C was continuously circulated through the organ chamber and a reservoir (total volume = 100 ml). The vessels were pressurized to 20 mm Hg in a closed, no-flow state with two burette manometers filled with Krebs buffer solution and maintained at the same level. With an inverted microscope (40-200x, Olympus IMT-2) connected to a video camera, the vessel image was projected onto a black and white television monitor (Panasonic, Japan). A video electronic dimension analyzer (Living Systems Instrumentation, Burlington, Vt.) was used to measure internal lumen diameter. A pressure transducer measured distending pressure via a sidearm cannula immediately proximal to one of the micropipettes. Measurements were recorded with a strip-chart recorder (Western Graphtec, Irvine, Calif.) (Fig. 1).
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In selected experiments, the cyclooxygenase inhibitor indomethacin (10 µmol/L) or the nitric oxide synthase inhibitor N G-methyl-L-arginine (L-NMMA) (30µmol/L) was added to the organ chamber reservoir at least 15 minutes before the administration of protamine. Only one of these blocking agents was used for each vessel.
Endothelial denudation
For the role of the endothelium in modulating vasomotor reactivity to be determined, selected control vessels were denuded of endothelium by abrading the luminal surface with a human hair and then flushing the lumen with air bubbles and Krebs buffer solution. The vessels were mounted and responses to protamine sulfate, heparin, protamine in the presence of heparin, histamine, and sodium nitroprusside were examined by means of the methods described herein.
Drugs
Histamine, the thromboxane A2 analog U46619, sodium nitroprusside, indomethacin, and L-NMMA were obtained from Sigma Chemical Co. (St. Louis, Mo.). Protamine sulfate was obtained from Lyphomed (Deerfield, Ill.). Heparin was obtained from Elkins-Sinn, Inc. (Cherry Hill, N.J.). Protamine, heparin, histamine, sodium nitroprusside, and L-NMMA were dissolved in ultrapure distilled water. U46619 was dissolved in ethanol to made a 10 mmol/L stock solution. Indomethacin was dissolved in minimal ethanol and water to make a 20 mmol/L stock solution. All stock solutions were stored at -20° C. All dilutions were prepared daily.
Data analysis
Microvascular responses are expressed as the percent relaxation of the U46619-induced constriction. Values are expressed as mean ± standard error of the mean. No more than one dose-response intervention with each drug was examined on vessels from each animal (n denotes number of animals from which vessels were examined). Responses of vessels to each drug were compared by two-way analysis of variance with repeated-measures design. Selected comparisons of responses at individual drug doses were performed with one-way analysis of variance and Scheffe's test for multiple comparisons. Statistical significance was assumed when p was less than 0.05.
RESULTS
Vessel characteristics
Pulmonary microvessels ranged from 90 to 190 µm in internal diameter, averaging 141 ± 10 µm, and 138 ± 6 µm, and 138 ± 6 µm in diameter in the control, partial CPB, and total CPB groups, respectively. The percent precontraction of vessels was 21% ± 1%, 22% ± 2%, and 22% ± 2% in the control, partial CPB, and total CPB groups, respectively. Similar concentrations of the thromboxane analog U46619 were required to attain these degrees of contraction in the groups.
Pulmonary vascular resistance
In the total CPB group, pulmonary vascular resistance increased substantially over the baseline resistance when normal pulmonary perfusion was reestablished. After 30 minutes of normal perfusion, pulmonary vascular resistance returned to near the baseline level. In contrast, pulmonary vascular resistance after reestablishment of normal pulmonary perfusion in the partial CPB group increased only modestly over the baseline level (Fig. 2).
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Histamine-induced relaxation of control microvessels was changed to a significant contractile response after denudation (see Fig. 6). The relaxation of pulmonary vessels to the endothelium-independent vasodilation to nitroprusside was slightly reduced after endothelial denudation, most likely because of damage to the vascular smooth muscle layer during the endothelial removal procedure.
DISCUSSION
This study shows that protamine causes endothelium-dependent relaxation of pulmonary microvessels. This relaxation response is significantly reduced after either total CPB or partial CPB. Histamine, which has been implicated in causing pulmonary hypertension during protamine infusion, 12 elicits endothelium-dependent relaxation of control pulmonary microvessels but produces contraction after total CPB.
Protamine may produce side effects that can complicate the outcome of an otherwise uneventful operation involving CPB. These side effects include pulmonary hypertension, 5,6 systemichypotension, 3,4 and left ventricular dysfunction. Although some studies suggest a direct effect of protamine on myocardial function, 13,14 it is more likely that the major detrimental effect of protamine on pulmonary perfusion and cardiac function is due to direct and indirect influences on vascular tone.
The mechanisms involved in the protamine-induced pulmonary vasoconstriction after heparinization are not clear. Some investigators have suggested that the release of thromboxane A2 after administration of protamine to heparinized animals is responsible for the pulmonary vasoconstriction. 15,16 This is consistent with the present study at low to moderate concentrations of protamine, because cyclooxygenase inhibition with the addition of indomethacin to the organ chamber resulted in reduced microvascular contractile responses (see Fig. 7). At higher concentrations, protamine appears to cause the release of a vasodilation prostaglandin substance, because the addition of indomethacin caused a reduced vasodilator response to a 100 µmol/L protamine concentration. Others have suggested that the activation of the complement system through the classic pathway by protamine-heparin interaction 17 or release of histamine from the lung tissue after protamine injection to the right atrium 12 is a cause of increased pulmonary vascular resistance. When protamine was introduced into the ascending aorta 18 or into the left atrium, 12 no changes were seen in either systemic or pulmonary vascular resistance. In addition, slight increases in blood pressure and in cardiac output were observed. This suggests that the passage of protamine through the lungs may be directly associated with the side effects appearing after protamine administration. Thus it is believed that alterations in pulmonary vessel reactivity may have a significant influence on the detrimental response to protamine after cardiac operations.
Previous studies on the adverse effects of protamine infusion on hemodynamics have reported these effects to be independent of the presence or absence of heparin. 13 However, Hird and coworkers 14 recently reported that protamine alone, but not heparin alone or protamine bound to heparin, reduces myocyte contractile function and ß-adrenergic responsiveness. In the present study, vascular regulation rather than myocardial contractile function was examined. Control pulmonary microvessels in the presence of heparin showed no significant difference in the response to protamine compared with the response in the absence of heparin. This same pattern was evident in microvessels from the total CPB group, in that vessels exposed to either protamine alone or protamine in the presence of heparin demonstrated reduced and similar relaxations. These findings indicate that in the pulmonary microcirculation, protamine alone has vascular effects similar to those of the protamine-heparin complex or that protamine itself and not protamine-heparin complex is responsible for these changes.
Pulmonary vascular endothelium has the ability to modulate pulmonary vascular resistance and pulmonary blood pressure. 19-21 Nitric oxide, which is the biologically active component of the endothelium-derived relaxation factor, 8 can cause pulmonary vasodilatation. A recent study suggests that protamine can cause the release of endothelium-derived nitric oxide from arterial endothelium. 10 This might contribute to protamine-induced systemic hypotension during protamine infusion. Total CPB and pulmonary ischemia can cause changes in endothelium-dependent pulmonary microvascular responses. 1 In the present study, we determined that the vasoactive responses to heparin, protamine, and protamine-heparin complex are all modulated by the endothelium. It is likely, therefore, that the altered responses of pulmonary microvessels to these substances after CPB are at least partially related to altered endothelial function and the consequent reduced vasodilation effects of these agents.
Pulmonary microvessels less then 190 µm in diameter were examined in the present study. Inasmuch as these vessels provide the majority of resistance to pulmonary flow, 11 this may provide more clinically relevant information regarding the changes in pulmonary vascular reactivity during CPB than studies examining rings or strips of large pulmonary arteries.
The present study suggests that the interuption of pulmonary perfusion during extracorporeal circulation may alter pulmonary vascular reactivity, contribute to post-CPB pulmonary hypertension, and cause detrimental effects on lung function. These findings may have implications regarding the preservation of pulmonary tissue during CPB and the increase in pulmonary vascular resistance frequently observed during neutralization of heparin during cardiac operations.
Footnotes
References
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p < 0.05 versus control at denoted concentration.