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Altered Reactivity to Acetylcholine in the Pulmonary Circulation After Cardiopulmonary Bypass Is Part of Reperfusion Injury
Original Contributions Altered Reactivity to Acetylcholine in the Pulmonary Circulation After Cardiopulmonary Bypass Is Part of Reperfusion Injury Monika Angdin, MD,* Goran Settergren, MD, PhD,* Raphael Astudillo, MD,† Jan Liska, MD, PhD† Department of Surgical Sciences, Karolinska Institute, Stockholm, Karolinska Hospital, Stockholm, Sweden.
*Associate Professor of Anaesthetics, Division of Cardiothoracic Anesthetics and Intensive Care †Assistant Professor of Thoracic Surgery, Division of Thoracic Surgery Address correspondence to Dr. Angdin at the Department of Cardiothoracic Anaesthetics and Intensive Care, Karolinska Hospital, S–171 76 Stockholm, Sweden. Supported by a grant from the Karolinska Institute, Stockholm, Sweden. Received for publication June 24, 1997; revised manuscript accepted for publication October 24, 1997.
Study Objective: To investigate whether a time sequence of acetylcholine (ACH) reactivity indicative of endothelial reperfusion injury could be demonstrated in the pulmonary circulation in patients after cardiopulmonary bypass (CPB). Design: Prospective study. Setting: Operating theater and intensive care unit of a university hospital. Patients: 10 ASA physical status III and IV patients with ischemic or valvular heart disease. Interventions: Pulmonary vascular resistance index (PVRI) was measured before and during an infusion of ACH. This procedure was done after induction of anesthesia but before surgery, immediately after weaning from bypass, and at 1 to 1.5 and 4 hours after CPB. Measurements and Main Results: ACH caused a decrease in PVRI before (p ,0.01) and directly after CPB (p ,0.05) but not at 1 to 1.5 or 4 hours after bypass. Conclusions: The maintained reactivity to ACH directly after CPB, followed by no reaction at 1 to 1.5 and 4 hours, was in agreement with experimental findings and indicates endothelial reperfusion injury caused by the period with no blood flow through the pulmonary artery during CPB and subsequent reperfusion. © 1998 by Elsevier Science Inc. Keywords: Acetylcholine (ACH); cardiopulmonary bypass (CPB); circulation, pulmonary; endothelium-derived relaxing factor (EDRF); pulmonary vascular resistance; reperfusion injury.
Introduction The concept of reperfusion injury or ischemia-reperfusion injury is based on the observation that the functional or structural cell injury, which occurs after a limited period without blood flow, is in some respects not manifested directly after the ischemic period, when reperfusions starts, but becomes evident first after a period of reperfusion. One of the earliest signs of reperfusion injury
Journal of Clinical Anesthesia 10:126 –132, 1998 © 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0952-8180/98/$19.00 PII S0952-8180(97)00256-0
ACH reactivity in pulmonary circulation post-CPB: Angdin et al.
seems to be a diminished production of nitric oxide (NO) from the endothelium, both the basal release1– 4 and the receptor stimulated release.1,5 The endothelium normally protects against vasospasm and thrombosis by the production of NO, which is the active component of endothelium-derived relaxing factor (EDRF),6 and prostacyclin. Through a synergistic action, these two substances relax vascular smooth muscle, antagonize the production and activity of endothelin-1, inhibit the adhesion and aggregation of platelets, and promote platelet disaggregation.7 NO also has been shown to inhibit chemotaxis, activation and adherence to the endothelium of polymorphonuclear leukocytes (PMN), all of which play an important role in reperfusion injury.2,3 Normally, blood vessels react with a dose-dependent vasorelaxation to an infusion of acetylcholine (ACH),8 which is an endothelium dependent vasodilator, through the L-arginine–NO pathway.9 After ischemia and a period of reperfusion this reaction is attenuated or lost.1,5 The time interval before this agonist release of NO is diminished varies in different studies, from a few minutes in the cat heart5 up to 90 minutes in the pulmonary circulation of the rabbit.1 This impairment of endothelium-dependent relaxation is thought to be at least partially caused by the production of free radicals during reperfusion.1,4,5 Oxygen-derived radicals seem to selectively impair receptor-dependent NO production by the endothelium.10 In cardiac operations in which cardiopulmonary bypass (CPB) is used there is almost no blood flow through the pulmonary artery during the period on bypass, because blood coming to the right atrium is drained to the extracorporeal circuit. It has been shown, both in children11 and adults,12 that the reactivity to ACH in the pulmonary circulation was attenuated or lost after CPB. This was interpreted as endothelial dysfunction since endothelium-independent vasodilators such as NO or sodium nitroprusside continued to decrease the pulmonary vascular resistance (PVR) after CPB. In Davenpeck et al.’s study1 in rabbits, the ACH-reactivity in the pulmonary artery, when studied in vitro, was intact directly after a period of ischemia caused by clamping of the pulmonary artery of one lung in vivo. However, after a similar period of ischemia followed by reperfusion in vivo, the ACH-reactivity measured in vitro deteriorated, thus indicating reperfusion injury. This attenuated reaction first became significant after 90 minutes of reperfusion. The current study was designed to investigate whether reperfusion injury also could be demonstrated in vivo in humans after CPB. The ACH-reactivity was tested before CPB and 1 to 1.5 and 4 hours after CPB, but also directly after CPB and before the administration of protamine. The period of partial bypass with flow through the pulmonary artery and an ejecting ventricle intentionally was made very short, and all blood continued to be drained from the right atrium to the extracorporeal circuit until 2 to 3 minutes before weaning from bypass.
Materials and Methods With approval from the Ethical Committee of Karolinska Hospital and informed patient consent, ten adult ASA physical status III and IV patients with ischemic or mitral heart disease, who reacted with a decrease in PVRI after an infusion of ACH, were investigated. The aim was to include patients with a moderately elevated PVRI secondary to severe ischemic heart disease or valvular dysfunction. Four patients were excluded who did not react to an infusion of ACH with a lowered PVRI before surgery. Ketobemidone 5 to 7.5 mg was used intramuscularly (IM) as premedication 1 hour before induction of anesthesia. An arterial catheter was inserted in the left radial artery before anesthesia. The techniques of anesthesia and CPB were standardized. Anesthesia was induced with midazolam 3 to 5 mg and fentanyl 0.5 to 1 mg followed by pancuronium (coronary artery surgery) or vecuronium (valve replacement) 0.1 mg/kg for muscular relaxation. For maintenance of anesthesia, a continuous infusion of fentanyl (4 to 5 mg/kg/hr) and midazolam (40 to 50 mg/kg/hr) was used throughout the operation. Ketobemidone 0.5 to 2 mg/hr in a continuous intravenous (IV) infusion was used for postoperative analgesia, and propofol 50 to 100 mg/hr was given for sedation. Neither nitroglycerin nor nitroprusside was used during the study periods. In addition, they were always discontinued at least 30 minutes before measurements were performed. The patients remained on controlled mechanical ventilation during the study period. After induction of anesthesia, a Swan-Ganz catheter (Edwards, 7.5 Fr, Irvine CA, USA) was inserted through the right internal jugular vein. Prior to the start of CPB 300 IU/kg of heparin were administered. The technique of CPB included nonpulsatile flow with a roller pump, crystalloid priming solution, and a membrane oxygenator. Patients’ temperatures were lowered to 34°C after starting CPB and re-warmed to a rectal temperature of 36°C before weaning from bypass. Cardiac output (CO) was measured as pulmonary artery flow by the thermodilution technique (Baxter COM-2, Edwards Critical Care Division, Irvine CA, USA) using 10 ml of iced 5% glucose solution. Two separate pairs of injections, randomly distributed in relation to the respiratory cycle, were performed. Pressure measurements were made using transducers (Abbott, SR 88659 SDT, No. Chicago, IL). The transpulmonary pressure gradient (TPPG) was determined in duplicate from mean pulmonary artery pressure (MPAP) and pulmonary capillary wedge pressure (PCWP) before bypass, and from left atrial (LA) pressure after bypass. PVRI was calculated as the TPPG divided by cardiac index [CI 5 CO/body surface area (m2 BSA)]. The infusion rate of 1 mg/ml solution of ACH (Miocholt, OMY Pharmaceuticals, San Germa´n, Puerto Rico) given through the Swan-Ganz catheter into the right ventricle was calculated from CO, to provide a 1026 mol/L concentration in the pulmonary artery. With a CO of 5 L/min, the infusion rate was 55 ml/hr. Oxygenation was monitored with pulse oximetry (Ultima, Datex, Helsinki, Finland). In six patients, arterial blood J. Clin. Anesth., vol. 10, March 1998
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Original Contributions
Table 1. Demographic Data of the Ten Patients Included in the Study Pt. No.
Type of Surgery
Age (yrs)
Gender
MPAP (baseline)
PVRI (baseline)
CPB Time
AO Time
1 2 3 4 5 6 7 8 9 10
CABG CABG MVR CABG MVR CABG CABG MVR CABG CABG Mean
69 75 58 71 53 67 70 63 68 77 67
F F F M F F M F M F
21 18 20 14 28 22 19 13 25 32 21
404 566 233 241 411 349 261 231 119 307 312
124 94 107 79 89 76 64 78 127 96 93
64 47 65 45 65 33 30 43 68 50 51
MPAP 5 mean pulmonary artery pressure; PVRI 5 pulmonary vascular resistance index, CPB time 5 time on cardiopulmonary bypass; AO time 5 time with aortic occlusion; CABG 5 coronary artery bypass grafting; MVR 5 mitral valve replacement.
gases were taken before and during ACH infusion (ABL, Radiometer, Copenhagen, Denmark). PVRI was determined in duplicate, before starting the ACH infusion, and after 5 minutes of infusion. This procedure was done at the following times: (1) after induction of anesthesia, before surgery, (2) directly after weaning from CPB before protamine was administered, and (3) 1 to 1.5 hours, and (4) 4 hours after CPB. Total bypass was maintained up to the end of CPB in order to minimize the time with blood flow through the pulmonary artery before the first measurement after CPB.
Statistical Analysis To evaluate the effect of ACH, a two-way analysis of variance (ANOVA) was performed, followed by planned comparisons corrected for repeated measurements. A p-value less than 0.05 was considered significant. The variation of a single determination was calculated from double determinations using the formula s 5 =(¥ d2)/2n, where s is the variation, d is the difference between double determinations, and n is the number of double determinations.
Results Demographic data of the ten patients included in the study are presented in Table 1. PVRI, TPPG, and CI are shown in Figure 1. Reductions of PVRI were seen before surgery and at the measurement directly after weaning from bypass. There were no reactions at 1 to 1.5 and 4 hours after CPB. PVRI was increased at the last two measurements (3 and 4) compared with measurements 1 and 2, (i.e., before and directly after CPB). All hemodynamic parameters are shown in Table 2. Mean aortic occlusion time was 51 minutes (range 30 to 68 min) and the duration of CPB was 93 minutes (64 to 127 min). The variation of a double determination was 43 dyn 3 sec/cm5/m2 for PVRI (13%), 0.9 mmHg for the TPPG (10%), and 0.14 L/min/m2 for CI (7%). 128
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Discussion The maintained reaction with pulmonary vasodilatation during the infusion of ACH directly after weaning from CPB (i.e., after no more than 5 to 10 minutes of reperfusion, followed by no reaction at 1 to 1.5 and 4 hours after CPB) was consistent with the concept of ischemia-reperfusion injury. The time course was similar to that observed experimentally.1 The development of reperfusion injury seems to take longer time in the lung1 than in the heart,5 probably due to the unique blood supply of the lung from two different sources (i.e., the bronchial and pulmonary arteries) and also due to the fact that the lungs have an oxygen supply via the alveoli, which is independent of the circulation. One of the earliest manifestations of ischemia-reperfusion injury appears to be a dysfunction of the vascular endothelium, probably due to the generation of oxygenderived free radicals. These are in part produced by the endothelium itself, but adherent neutrophils also are likely implicated.7,13,14 It seems to take approximately 20 minutes of reperfusion before there is a significant increase in PMN adherence to the endothelium.2 The adherence and activation of neutrophils to the endothelium leads to a number of negative events.7 In the current study, only the endothelium-dependent vasodilatation was analyzed. Experimentally it has been shown that basal NO release is decreased after ischemia-reperfusion, both in the pulmonary circulation1,4 and the heart.2,3 In the pulmonary vasculature, this seem to happen even earlier than the decrease of NO released by ACH.1 In one study in piglets, it was shown that the basal NO production is reduced by 70% in the pulmonary circulation after CPB.4 A decreased basal NO release could be one factor explaining the higher levels of PVRI at 1 to 1.5 and 4 hours after CPB in the current study. In another study in pigs,15 L-NAME, which is an inhibitor of endothelial NO-synthase,9 was administered 15 minutes after CPB. The resulting changes in CO and pulmonary artery pressure were interpreted as a maintained basal NO release in the pulmonary circulation after CPB and a maintained NO synthase function. In
ACH reactivity in pulmonary circulation post-CPB: Angdin et al.
Figure 1. Changes in pulmonary vascular resistance index (PVRI), transpulmonary pressure difference (TPPD), and cardiac index (CI) (means 6 SE) in ten patients undergoing open-heart surgery investigated before and during acetylcholine (ACH) infusions. Measurements were performed before surgery, directly after weaning from cardiopulmonary bypass (CPB), and at 1 to 1.5 and 4 hours after CPB. *p ,0.05, **p ,0.01 (statistically significant differences).
the same study, a decreased reaction of PVR to ACH after CPB was also demonstrated, which was in agreement with human studies.11,12
Elevated levels of endogenous catecholamines after CPB may further affect PVR.16 Another explanation for the increased PVR after CPB might be increased producJ. Clin. Anesth., vol. 10, March 1998
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Note: Data are means 6 SE. Hemodynamic variables are presented in ten patients before the start of operation, immediately after weaning from cardiopulmonary bypass (CPB), and at 1 to 1.5 and 4 hours after CPB. ACH 5 acetylcholine, MAP 5 mean arterial pressure, TPPD 5 transpulmonary pressure difference, MPAP 5 mean pulmonary artery pressure, PCWP 5 pulmonary capillary wedge pressure, LAP 5 left atrial pressure (mmHg), CI 5 cardiac index (l/min/m2), PVRI 5 pulmonary vascular resistance index (dyn 3 sec/cm5/m2), HR 5 heart rate (beats/min), PaO2, PACO (kPa) and pH. * 5 6 patients, n.s. 5 nonsignificant.
n.s. n.s. n.s. n.s. 75 6 3 105 6 8 36 6 3 7.42 6 0.00 75 6 3 105 6 8 38 6 4 7.41 6 0.00 n.s. n.s. n.s. n.s. 69 6 3 150 6 22 38 6 1 7.39 6 0.00 69 6 3 135 6 22 38 6 1 7.40 6 0.01 n.s. n.s. n.s. n.s. 66 6 3 232 6 60 37 6 1 7.43 6 0.01 69 6 3 240 6 68 34 6 1 7.42 6 0.01 54 6 2 188 6 38 35 6 2 7.44 6 0.00 56 6 3 210 6 38 36 6 2 7.43 6 0.00
n.s. n.s. n.s. n.s.
n.s. n.s. n.s. n.s. n.s. n.s. 82 6 4 10 6 1 23 6 1 13 6 1 2.1 6 0.1 384 6 39 80 6 5 10 6 1 23 6 2 12 6 1 2.0 6 0.1 433 6 44 n.s. n.s. n.s. n.s. n.s. n.s. 77 6 5 961 20 6 1 11 6 1 1.9 6 0.1 389 6 48 79 6 4 961 21 6 1 12 6 1 2.0 6 0.1 384 6 54 n.s. ,0.01 n.s. n.s. n.s. ,0.05 73 6 3 761 17 6 1 10 6 1 2.4 6 0.1 238 6 27 64 6 2 861 17 6 1 961 2.5 6 0.1 274 6 30 78 6 5 661 19 6 2 13 6 2 2.0 6 0.1 254 6 35
MAP (mmHg) TPPD (mmHg) MPAP (mmHg) PCWP/LAP (mmHg) CI (L/min/m2) PVRI (dynes z sec z m25 z m22) HR (bpm) PaO2* (mmHg) PaCO2* (mmHg) pH*
80 6 6 861 21 6 2 13 6 2 2.1 6 0.1 312 6 38
n.s. ,0.01 n.s. n.s. n.s. ,0.01
ACH Control p-value ACH Control ACH ACH Control
Table 2. Hemodynamic Variables
Before CPB
p-value
Control
After CPB
p-value
1 to 1.5 Hours After CPB
4 Hours After CPB
p-value
Original Contributions
tion of other vasoconstrictors such as endothelin17,18 and thromboxane A2.19 Changes in PVR over prolonged periods of time during anesthesia and surgery with CPB must be interpreted with great caution, because there are many factors that have an influence on PVR. When comparing PVR before and after CPB, one must consider changes in blood viscosity due to the reduction in hematocrit (Hct), which normally occurs during CPB due to dilution of the blood with priming solution of the extracorporeal circuit. Normally, this reduced Hct is slowly normalized during the first 4 hours after bypass.20 Thus, the PVR level directly after CPB, which was similar to that before surgery, could be due to a reduced blood viscosity combined with an increase in vascular tone. However, blood viscosity was not measured or calculated in the present study. When investigating the effect of ACH on PVRI, it is important that other determinants of PVRI, such as PaCO2, pHa, and PaO2, are stable during the infusion. Usually this is no problem during continuous ventilator treatment with fixed ventilator settings. Directly after the transition from oxygenator to lung ventilation, during weaning from CPB, blood gases could be unstable. However, in the current study these parameters did not change. With intact endothelial function, the expected effect of ACH on PaO2 is a decrease, but for some reason this was not observed. A methodologic difference between the current study and the experimental study of the ACH reactivity of the endothelium in vitro1 should also be noted. The rabbits were ventilated with an endotracheal tube and for this reason both lungs were ventilated also during the ischemic period, when the pulmonary artery on one side was clamped. In the current study, the systemic blood flow was oxygenated in the extracorporeal circuit and, following our normal clinical routine, the lungs were left without ventilation until partial bypass was started shortly before weaning from bypass. It is difficult to know what importance—if any—this difference in methodology could have. Obviously, the maintained ventilation of the lung, when the pulmonary artery was clamped, did not prevent the development of endothelial dysfunction. In the current study, four patients were excluded because they did not react to an infusion of ACH before operation. It has been shown that the reaction to ACH in the pulmonary artery depends on the preexisting vascular tone. Patients with low pulmonary artery pressure and almost totally dilated vascular beds, as well as those with very high long-standing pulmonary hypertension, do not react to infusion of ACH.21,22 A number of other factors such as hypercholesterolemia,23 arteriosclerosis,24 uremia,25 diabetes,9,26 chronic hypoxia,27 and hypertension9,22,28 also influence this pathway and attenuate the response to an infusion of ACH. Before starting the study, we had evaluated the response of different concentrations of ACH and found the best response with 1026 M concentration. This concentration is in agreement with many other studies, both animal and human in vivo and in vitro studies, where the maximal vasodilatation is achieved at 1026 M concentration of ACH.1,11,22,23,26,27 A study by Evora et al.29 has shown that protamine sulfate produces
ACH reactivity in pulmonary circulation post-CPB: Angdin et al.
concentration-dependent relaxation of canine pulmonary artery segments to a significantly greater level in vessels with intact endothelium compared with segments without endothelium. They also showed that a heparin-protamine complex failed to induce vasodilatation. Another study, by Akata et al.,30 has shown that protamine causes an impairment of endothelium-dependent relaxation induced by ACH. However, in the presence of heparin, the reaction to ACH was normal. To avoid the effect of protamine, we did the measurement after bypass, before protamine was administered. The lungs were once considered resistant to ischemia during CPB due to their unique blood and oxygen supply. It is now understood that the lungs are also subjected to ischemia reperfusion injury in situations where there is no blood flow through the pulmonary artery. In an earlier study, we demonstrated a reversible functional impairment of the pulmonary artery endothelium in humans after 105 minutes of CPB.12 In an experimental study in pigs, where there was no flow through the pulmonary artery for 18 hours, while the systemic circulation was maintained with venoarterial bypass and the heart was beating, the result was severe lung damage leading to a mortality of 100%, when the animals were weaned from bypass.31 This observation stresses the importance of maintaining flow through the pulmonary artery in patients during treatment with extracorporeal membrane oxygenation and trying to make the period with total bypass as short as possible during open-heart surgery, especially during long procedures. A period with partial bypass before weaning is also beneficial for the heart, because it helps to normalize an abnormally high left ventricular compliance after the period with total bypass, as was recently demonstrated.32 In conclusion, the result of the current study supports the concept that altered reactivity to ACH in the pulmonary circulation after CPB is reperfusion injury.
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Acknowledgments This study was supported with a grant from the Karolinska Institute, Stockholm, Sweden. The authors also thank Elisabeth Berg for statistical advice.
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Pulmonary Artery Endothelial Dysfunction Following Ischemia and Reperfusion of the Rabbit Lung Kelly L. Davenpeck, Jin-ping Guo, Allan M. Lefer Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA. Abstract We studied endothelial dysfunction of the rabbit pulmonary artery following in vivo ischemia and reperfusion of the lung, and also investigated the mechanisms of endothelium-dependent relaxation in these arteries. Intrapulmonary arteries were isolated from rabbits subjected to ischemia and reperfusion of one lung. Percent relaxation values of sham-operated (i.e. nonischemic) pulmonary arteries to endothelium-dependent vasodilators acetylcholine (ACh) and A23187 were 72 6 4 and 65 6 4%, respectivley, while relaxation to the endothelium-dependent dilator NaNO2 was 97 61%. The relaxation of control artery rings to ACh and A23187 were significantly decreased to 2 6 1 and 5 6 4%, respectively, following addition of N-omega-nitro-Larginine methyl ester, while relaxation following treatment with indomethacin or glybenclamide remained normal. Relaxation of NaNO2 was not altered by pretreatment with any of the above compounds. Thus, pulmonary artery relaxation to the endothelium-dependent dilators ACh and A23187 appears to be mediated by the release of EDRF. Endothelium-dependent relaxation of pulmonary arteries from lungs exposed to 90 min of ischemia and 30 min of reperfusion remained essentially normal, while 90 min of ischemia followed by 60 min of reperfusion resulted in a significant decrease in endothelium-dependent relaxation to A23187 to 37 6 7% (p , 0.05), whereas the response to ACh was reduced only to 57 6 3% (not significant). 90 min of ischemia followed by 90 min of reperfusion resulted in significant attenuation of endotheliumdependent relaxation to both ACh (36 6 4%) and A23187 (33 6 7%). Intrapulmonary artery rings from rabbits given superoxide dismutase starting 15 min before reperfusion showed an improved relaxation to ACh and A23187 (55 6 5 and 58 6 11%, respectivley, at 90 min of reperfusion). Thus, in vivo ischemia and reperfusion of the rabbit lung results in significant endothelial dysfunction, which worsens with increasing reperfusion time. Some of the endothelial dysfunction appears to be related to superoxide radical formation. Reprinted from the Journal of Vascular Research 1993;30:145–53.
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*Associate Professor of Anaesthetics, Division of Cardiothoracic Anesthetics and Intensive Care †Assistant Professor of Thoracic Surgery, Division of Thoracic Surgery Address correspondence to Dr. Angdin at the Department of Cardiothoracic Anaesthetics and Intensive Care, Karolinska Hospital, S–171 76 Stockholm, Sweden. Supported by a grant from the Karolinska Institute, Stockholm, Sweden. Received for publication June 24, 1997; revised manuscript accepted for publication October 24, 1997.
Study Objective: To investigate whether a time sequence of acetylcholine (ACH) reactivity indicative of endothelial reperfusion injury could be demonstrated in the pulmonary circulation in patients after cardiopulmonary bypass (CPB). Design: Prospective study. Setting: Operating theater and intensive care unit of a university hospital. Patients: 10 ASA physical status III and IV patients with ischemic or valvular heart disease. Interventions: Pulmonary vascular resistance index (PVRI) was measured before and during an infusion of ACH. This procedure was done after induction of anesthesia but before surgery, immediately after weaning from bypass, and at 1 to 1.5 and 4 hours after CPB. Measurements and Main Results: ACH caused a decrease in PVRI before (p ,0.01) and directly after CPB (p ,0.05) but not at 1 to 1.5 or 4 hours after bypass. Conclusions: The maintained reactivity to ACH directly after CPB, followed by no reaction at 1 to 1.5 and 4 hours, was in agreement with experimental findings and indicates endothelial reperfusion injury caused by the period with no blood flow through the pulmonary artery during CPB and subsequent reperfusion. © 1998 by Elsevier Science Inc. Keywords: Acetylcholine (ACH); cardiopulmonary bypass (CPB); circulation, pulmonary; endothelium-derived relaxing factor (EDRF); pulmonary vascular resistance; reperfusion injury.
Introduction The concept of reperfusion injury or ischemia-reperfusion injury is based on the observation that the functional or structural cell injury, which occurs after a limited period without blood flow, is in some respects not manifested directly after the ischemic period, when reperfusions starts, but becomes evident first after a period of reperfusion. One of the earliest signs of reperfusion injury
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0952-8180/98/$19.00 PII S0952-8180(97)00256-0
ACH reactivity in pulmonary circulation post-CPB: Angdin et al.
seems to be a diminished production of nitric oxide (NO) from the endothelium, both the basal release1– 4 and the receptor stimulated release.1,5 The endothelium normally protects against vasospasm and thrombosis by the production of NO, which is the active component of endothelium-derived relaxing factor (EDRF),6 and prostacyclin. Through a synergistic action, these two substances relax vascular smooth muscle, antagonize the production and activity of endothelin-1, inhibit the adhesion and aggregation of platelets, and promote platelet disaggregation.7 NO also has been shown to inhibit chemotaxis, activation and adherence to the endothelium of polymorphonuclear leukocytes (PMN), all of which play an important role in reperfusion injury.2,3 Normally, blood vessels react with a dose-dependent vasorelaxation to an infusion of acetylcholine (ACH),8 which is an endothelium dependent vasodilator, through the L-arginine–NO pathway.9 After ischemia and a period of reperfusion this reaction is attenuated or lost.1,5 The time interval before this agonist release of NO is diminished varies in different studies, from a few minutes in the cat heart5 up to 90 minutes in the pulmonary circulation of the rabbit.1 This impairment of endothelium-dependent relaxation is thought to be at least partially caused by the production of free radicals during reperfusion.1,4,5 Oxygen-derived radicals seem to selectively impair receptor-dependent NO production by the endothelium.10 In cardiac operations in which cardiopulmonary bypass (CPB) is used there is almost no blood flow through the pulmonary artery during the period on bypass, because blood coming to the right atrium is drained to the extracorporeal circuit. It has been shown, both in children11 and adults,12 that the reactivity to ACH in the pulmonary circulation was attenuated or lost after CPB. This was interpreted as endothelial dysfunction since endothelium-independent vasodilators such as NO or sodium nitroprusside continued to decrease the pulmonary vascular resistance (PVR) after CPB. In Davenpeck et al.’s study1 in rabbits, the ACH-reactivity in the pulmonary artery, when studied in vitro, was intact directly after a period of ischemia caused by clamping of the pulmonary artery of one lung in vivo. However, after a similar period of ischemia followed by reperfusion in vivo, the ACH-reactivity measured in vitro deteriorated, thus indicating reperfusion injury. This attenuated reaction first became significant after 90 minutes of reperfusion. The current study was designed to investigate whether reperfusion injury also could be demonstrated in vivo in humans after CPB. The ACH-reactivity was tested before CPB and 1 to 1.5 and 4 hours after CPB, but also directly after CPB and before the administration of protamine. The period of partial bypass with flow through the pulmonary artery and an ejecting ventricle intentionally was made very short, and all blood continued to be drained from the right atrium to the extracorporeal circuit until 2 to 3 minutes before weaning from bypass.
Materials and Methods With approval from the Ethical Committee of Karolinska Hospital and informed patient consent, ten adult ASA physical status III and IV patients with ischemic or mitral heart disease, who reacted with a decrease in PVRI after an infusion of ACH, were investigated. The aim was to include patients with a moderately elevated PVRI secondary to severe ischemic heart disease or valvular dysfunction. Four patients were excluded who did not react to an infusion of ACH with a lowered PVRI before surgery. Ketobemidone 5 to 7.5 mg was used intramuscularly (IM) as premedication 1 hour before induction of anesthesia. An arterial catheter was inserted in the left radial artery before anesthesia. The techniques of anesthesia and CPB were standardized. Anesthesia was induced with midazolam 3 to 5 mg and fentanyl 0.5 to 1 mg followed by pancuronium (coronary artery surgery) or vecuronium (valve replacement) 0.1 mg/kg for muscular relaxation. For maintenance of anesthesia, a continuous infusion of fentanyl (4 to 5 mg/kg/hr) and midazolam (40 to 50 mg/kg/hr) was used throughout the operation. Ketobemidone 0.5 to 2 mg/hr in a continuous intravenous (IV) infusion was used for postoperative analgesia, and propofol 50 to 100 mg/hr was given for sedation. Neither nitroglycerin nor nitroprusside was used during the study periods. In addition, they were always discontinued at least 30 minutes before measurements were performed. The patients remained on controlled mechanical ventilation during the study period. After induction of anesthesia, a Swan-Ganz catheter (Edwards, 7.5 Fr, Irvine CA, USA) was inserted through the right internal jugular vein. Prior to the start of CPB 300 IU/kg of heparin were administered. The technique of CPB included nonpulsatile flow with a roller pump, crystalloid priming solution, and a membrane oxygenator. Patients’ temperatures were lowered to 34°C after starting CPB and re-warmed to a rectal temperature of 36°C before weaning from bypass. Cardiac output (CO) was measured as pulmonary artery flow by the thermodilution technique (Baxter COM-2, Edwards Critical Care Division, Irvine CA, USA) using 10 ml of iced 5% glucose solution. Two separate pairs of injections, randomly distributed in relation to the respiratory cycle, were performed. Pressure measurements were made using transducers (Abbott, SR 88659 SDT, No. Chicago, IL). The transpulmonary pressure gradient (TPPG) was determined in duplicate from mean pulmonary artery pressure (MPAP) and pulmonary capillary wedge pressure (PCWP) before bypass, and from left atrial (LA) pressure after bypass. PVRI was calculated as the TPPG divided by cardiac index [CI 5 CO/body surface area (m2 BSA)]. The infusion rate of 1 mg/ml solution of ACH (Miocholt, OMY Pharmaceuticals, San Germa´n, Puerto Rico) given through the Swan-Ganz catheter into the right ventricle was calculated from CO, to provide a 1026 mol/L concentration in the pulmonary artery. With a CO of 5 L/min, the infusion rate was 55 ml/hr. Oxygenation was monitored with pulse oximetry (Ultima, Datex, Helsinki, Finland). In six patients, arterial blood J. Clin. Anesth., vol. 10, March 1998
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Original Contributions
Table 1. Demographic Data of the Ten Patients Included in the Study Pt. No.
Type of Surgery
Age (yrs)
Gender
MPAP (baseline)
PVRI (baseline)
CPB Time
AO Time
1 2 3 4 5 6 7 8 9 10
CABG CABG MVR CABG MVR CABG CABG MVR CABG CABG Mean
69 75 58 71 53 67 70 63 68 77 67
F F F M F F M F M F
21 18 20 14 28 22 19 13 25 32 21
404 566 233 241 411 349 261 231 119 307 312
124 94 107 79 89 76 64 78 127 96 93
64 47 65 45 65 33 30 43 68 50 51
MPAP 5 mean pulmonary artery pressure; PVRI 5 pulmonary vascular resistance index, CPB time 5 time on cardiopulmonary bypass; AO time 5 time with aortic occlusion; CABG 5 coronary artery bypass grafting; MVR 5 mitral valve replacement.
gases were taken before and during ACH infusion (ABL, Radiometer, Copenhagen, Denmark). PVRI was determined in duplicate, before starting the ACH infusion, and after 5 minutes of infusion. This procedure was done at the following times: (1) after induction of anesthesia, before surgery, (2) directly after weaning from CPB before protamine was administered, and (3) 1 to 1.5 hours, and (4) 4 hours after CPB. Total bypass was maintained up to the end of CPB in order to minimize the time with blood flow through the pulmonary artery before the first measurement after CPB.
Statistical Analysis To evaluate the effect of ACH, a two-way analysis of variance (ANOVA) was performed, followed by planned comparisons corrected for repeated measurements. A p-value less than 0.05 was considered significant. The variation of a single determination was calculated from double determinations using the formula s 5 =(¥ d2)/2n, where s is the variation, d is the difference between double determinations, and n is the number of double determinations.
Results Demographic data of the ten patients included in the study are presented in Table 1. PVRI, TPPG, and CI are shown in Figure 1. Reductions of PVRI were seen before surgery and at the measurement directly after weaning from bypass. There were no reactions at 1 to 1.5 and 4 hours after CPB. PVRI was increased at the last two measurements (3 and 4) compared with measurements 1 and 2, (i.e., before and directly after CPB). All hemodynamic parameters are shown in Table 2. Mean aortic occlusion time was 51 minutes (range 30 to 68 min) and the duration of CPB was 93 minutes (64 to 127 min). The variation of a double determination was 43 dyn 3 sec/cm5/m2 for PVRI (13%), 0.9 mmHg for the TPPG (10%), and 0.14 L/min/m2 for CI (7%). 128
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Discussion The maintained reaction with pulmonary vasodilatation during the infusion of ACH directly after weaning from CPB (i.e., after no more than 5 to 10 minutes of reperfusion, followed by no reaction at 1 to 1.5 and 4 hours after CPB) was consistent with the concept of ischemia-reperfusion injury. The time course was similar to that observed experimentally.1 The development of reperfusion injury seems to take longer time in the lung1 than in the heart,5 probably due to the unique blood supply of the lung from two different sources (i.e., the bronchial and pulmonary arteries) and also due to the fact that the lungs have an oxygen supply via the alveoli, which is independent of the circulation. One of the earliest manifestations of ischemia-reperfusion injury appears to be a dysfunction of the vascular endothelium, probably due to the generation of oxygenderived free radicals. These are in part produced by the endothelium itself, but adherent neutrophils also are likely implicated.7,13,14 It seems to take approximately 20 minutes of reperfusion before there is a significant increase in PMN adherence to the endothelium.2 The adherence and activation of neutrophils to the endothelium leads to a number of negative events.7 In the current study, only the endothelium-dependent vasodilatation was analyzed. Experimentally it has been shown that basal NO release is decreased after ischemia-reperfusion, both in the pulmonary circulation1,4 and the heart.2,3 In the pulmonary vasculature, this seem to happen even earlier than the decrease of NO released by ACH.1 In one study in piglets, it was shown that the basal NO production is reduced by 70% in the pulmonary circulation after CPB.4 A decreased basal NO release could be one factor explaining the higher levels of PVRI at 1 to 1.5 and 4 hours after CPB in the current study. In another study in pigs,15 L-NAME, which is an inhibitor of endothelial NO-synthase,9 was administered 15 minutes after CPB. The resulting changes in CO and pulmonary artery pressure were interpreted as a maintained basal NO release in the pulmonary circulation after CPB and a maintained NO synthase function. In
ACH reactivity in pulmonary circulation post-CPB: Angdin et al.
Figure 1. Changes in pulmonary vascular resistance index (PVRI), transpulmonary pressure difference (TPPD), and cardiac index (CI) (means 6 SE) in ten patients undergoing open-heart surgery investigated before and during acetylcholine (ACH) infusions. Measurements were performed before surgery, directly after weaning from cardiopulmonary bypass (CPB), and at 1 to 1.5 and 4 hours after CPB. *p ,0.05, **p ,0.01 (statistically significant differences).
the same study, a decreased reaction of PVR to ACH after CPB was also demonstrated, which was in agreement with human studies.11,12
Elevated levels of endogenous catecholamines after CPB may further affect PVR.16 Another explanation for the increased PVR after CPB might be increased producJ. Clin. Anesth., vol. 10, March 1998
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Note: Data are means 6 SE. Hemodynamic variables are presented in ten patients before the start of operation, immediately after weaning from cardiopulmonary bypass (CPB), and at 1 to 1.5 and 4 hours after CPB. ACH 5 acetylcholine, MAP 5 mean arterial pressure, TPPD 5 transpulmonary pressure difference, MPAP 5 mean pulmonary artery pressure, PCWP 5 pulmonary capillary wedge pressure, LAP 5 left atrial pressure (mmHg), CI 5 cardiac index (l/min/m2), PVRI 5 pulmonary vascular resistance index (dyn 3 sec/cm5/m2), HR 5 heart rate (beats/min), PaO2, PACO (kPa) and pH. * 5 6 patients, n.s. 5 nonsignificant.
n.s. n.s. n.s. n.s. 75 6 3 105 6 8 36 6 3 7.42 6 0.00 75 6 3 105 6 8 38 6 4 7.41 6 0.00 n.s. n.s. n.s. n.s. 69 6 3 150 6 22 38 6 1 7.39 6 0.00 69 6 3 135 6 22 38 6 1 7.40 6 0.01 n.s. n.s. n.s. n.s. 66 6 3 232 6 60 37 6 1 7.43 6 0.01 69 6 3 240 6 68 34 6 1 7.42 6 0.01 54 6 2 188 6 38 35 6 2 7.44 6 0.00 56 6 3 210 6 38 36 6 2 7.43 6 0.00
n.s. n.s. n.s. n.s.
n.s. n.s. n.s. n.s. n.s. n.s. 82 6 4 10 6 1 23 6 1 13 6 1 2.1 6 0.1 384 6 39 80 6 5 10 6 1 23 6 2 12 6 1 2.0 6 0.1 433 6 44 n.s. n.s. n.s. n.s. n.s. n.s. 77 6 5 961 20 6 1 11 6 1 1.9 6 0.1 389 6 48 79 6 4 961 21 6 1 12 6 1 2.0 6 0.1 384 6 54 n.s. ,0.01 n.s. n.s. n.s. ,0.05 73 6 3 761 17 6 1 10 6 1 2.4 6 0.1 238 6 27 64 6 2 861 17 6 1 961 2.5 6 0.1 274 6 30 78 6 5 661 19 6 2 13 6 2 2.0 6 0.1 254 6 35
MAP (mmHg) TPPD (mmHg) MPAP (mmHg) PCWP/LAP (mmHg) CI (L/min/m2) PVRI (dynes z sec z m25 z m22) HR (bpm) PaO2* (mmHg) PaCO2* (mmHg) pH*
80 6 6 861 21 6 2 13 6 2 2.1 6 0.1 312 6 38
n.s. ,0.01 n.s. n.s. n.s. ,0.01
ACH Control p-value ACH Control ACH ACH Control
Table 2. Hemodynamic Variables
Before CPB
p-value
Control
After CPB
p-value
1 to 1.5 Hours After CPB
4 Hours After CPB
p-value
Original Contributions
tion of other vasoconstrictors such as endothelin17,18 and thromboxane A2.19 Changes in PVR over prolonged periods of time during anesthesia and surgery with CPB must be interpreted with great caution, because there are many factors that have an influence on PVR. When comparing PVR before and after CPB, one must consider changes in blood viscosity due to the reduction in hematocrit (Hct), which normally occurs during CPB due to dilution of the blood with priming solution of the extracorporeal circuit. Normally, this reduced Hct is slowly normalized during the first 4 hours after bypass.20 Thus, the PVR level directly after CPB, which was similar to that before surgery, could be due to a reduced blood viscosity combined with an increase in vascular tone. However, blood viscosity was not measured or calculated in the present study. When investigating the effect of ACH on PVRI, it is important that other determinants of PVRI, such as PaCO2, pHa, and PaO2, are stable during the infusion. Usually this is no problem during continuous ventilator treatment with fixed ventilator settings. Directly after the transition from oxygenator to lung ventilation, during weaning from CPB, blood gases could be unstable. However, in the current study these parameters did not change. With intact endothelial function, the expected effect of ACH on PaO2 is a decrease, but for some reason this was not observed. A methodologic difference between the current study and the experimental study of the ACH reactivity of the endothelium in vitro1 should also be noted. The rabbits were ventilated with an endotracheal tube and for this reason both lungs were ventilated also during the ischemic period, when the pulmonary artery on one side was clamped. In the current study, the systemic blood flow was oxygenated in the extracorporeal circuit and, following our normal clinical routine, the lungs were left without ventilation until partial bypass was started shortly before weaning from bypass. It is difficult to know what importance—if any—this difference in methodology could have. Obviously, the maintained ventilation of the lung, when the pulmonary artery was clamped, did not prevent the development of endothelial dysfunction. In the current study, four patients were excluded because they did not react to an infusion of ACH before operation. It has been shown that the reaction to ACH in the pulmonary artery depends on the preexisting vascular tone. Patients with low pulmonary artery pressure and almost totally dilated vascular beds, as well as those with very high long-standing pulmonary hypertension, do not react to infusion of ACH.21,22 A number of other factors such as hypercholesterolemia,23 arteriosclerosis,24 uremia,25 diabetes,9,26 chronic hypoxia,27 and hypertension9,22,28 also influence this pathway and attenuate the response to an infusion of ACH. Before starting the study, we had evaluated the response of different concentrations of ACH and found the best response with 1026 M concentration. This concentration is in agreement with many other studies, both animal and human in vivo and in vitro studies, where the maximal vasodilatation is achieved at 1026 M concentration of ACH.1,11,22,23,26,27 A study by Evora et al.29 has shown that protamine sulfate produces
ACH reactivity in pulmonary circulation post-CPB: Angdin et al.
concentration-dependent relaxation of canine pulmonary artery segments to a significantly greater level in vessels with intact endothelium compared with segments without endothelium. They also showed that a heparin-protamine complex failed to induce vasodilatation. Another study, by Akata et al.,30 has shown that protamine causes an impairment of endothelium-dependent relaxation induced by ACH. However, in the presence of heparin, the reaction to ACH was normal. To avoid the effect of protamine, we did the measurement after bypass, before protamine was administered. The lungs were once considered resistant to ischemia during CPB due to their unique blood and oxygen supply. It is now understood that the lungs are also subjected to ischemia reperfusion injury in situations where there is no blood flow through the pulmonary artery. In an earlier study, we demonstrated a reversible functional impairment of the pulmonary artery endothelium in humans after 105 minutes of CPB.12 In an experimental study in pigs, where there was no flow through the pulmonary artery for 18 hours, while the systemic circulation was maintained with venoarterial bypass and the heart was beating, the result was severe lung damage leading to a mortality of 100%, when the animals were weaned from bypass.31 This observation stresses the importance of maintaining flow through the pulmonary artery in patients during treatment with extracorporeal membrane oxygenation and trying to make the period with total bypass as short as possible during open-heart surgery, especially during long procedures. A period with partial bypass before weaning is also beneficial for the heart, because it helps to normalize an abnormally high left ventricular compliance after the period with total bypass, as was recently demonstrated.32 In conclusion, the result of the current study supports the concept that altered reactivity to ACH in the pulmonary circulation after CPB is reperfusion injury.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Acknowledgments This study was supported with a grant from the Karolinska Institute, Stockholm, Sweden. The authors also thank Elisabeth Berg for statistical advice.
18.
19.
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Pulmonary Artery Endothelial Dysfunction Following Ischemia and Reperfusion of the Rabbit Lung Kelly L. Davenpeck, Jin-ping Guo, Allan M. Lefer Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA. Abstract We studied endothelial dysfunction of the rabbit pulmonary artery following in vivo ischemia and reperfusion of the lung, and also investigated the mechanisms of endothelium-dependent relaxation in these arteries. Intrapulmonary arteries were isolated from rabbits subjected to ischemia and reperfusion of one lung. Percent relaxation values of sham-operated (i.e. nonischemic) pulmonary arteries to endothelium-dependent vasodilators acetylcholine (ACh) and A23187 were 72 6 4 and 65 6 4%, respectivley, while relaxation to the endothelium-dependent dilator NaNO2 was 97 61%. The relaxation of control artery rings to ACh and A23187 were significantly decreased to 2 6 1 and 5 6 4%, respectively, following addition of N-omega-nitro-Larginine methyl ester, while relaxation following treatment with indomethacin or glybenclamide remained normal. Relaxation of NaNO2 was not altered by pretreatment with any of the above compounds. Thus, pulmonary artery relaxation to the endothelium-dependent dilators ACh and A23187 appears to be mediated by the release of EDRF. Endothelium-dependent relaxation of pulmonary arteries from lungs exposed to 90 min of ischemia and 30 min of reperfusion remained essentially normal, while 90 min of ischemia followed by 60 min of reperfusion resulted in a significant decrease in endothelium-dependent relaxation to A23187 to 37 6 7% (p , 0.05), whereas the response to ACh was reduced only to 57 6 3% (not significant). 90 min of ischemia followed by 90 min of reperfusion resulted in significant attenuation of endotheliumdependent relaxation to both ACh (36 6 4%) and A23187 (33 6 7%). Intrapulmonary artery rings from rabbits given superoxide dismutase starting 15 min before reperfusion showed an improved relaxation to ACh and A23187 (55 6 5 and 58 6 11%, respectivley, at 90 min of reperfusion). Thus, in vivo ischemia and reperfusion of the rabbit lung results in significant endothelial dysfunction, which worsens with increasing reperfusion time. Some of the endothelial dysfunction appears to be related to superoxide radical formation. Reprinted from the Journal of Vascular Research 1993;30:145–53.
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