Assessment of hepatic ischemia–reperfusion injury by simultaneous measurement of tissue pO2, pCO2, and pH

Microvascular Research 67 (2004) 38 – 47 www.elsevier.com/locate/ymvre

Assessment of hepatic ischemia–reperfusion injury by simultaneous measurement of tissue pO2, pCO2, and pH Dirk Uhlmann, a,* Uta-Carolin Pietsch, b Stefan Ludwig, a Jochen Hess, a Barbara Armann, a Gabor Gaebel, a Evelyn Escher, a Lutz Schaffranietz, b Andrea Tannapfel, c Martin Fiedler, d Johann Hauss, a and Helmut Witzigmann a a

2nd Department of Surgery, University of Leipzig, 04103 Leipzig, Germany b Department of Anesthesiology, University of Leipzig, Leipzig, Germany c Institute of Pathology, University of Leipzig, Leipzig, Germany d Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University of Leipzig, Germany Received 7 July 2003

Abstract Introduction: The objective of this study was to determine whether the simultaneous measurement of tissue pH, pCO2, and pO2 with a multiple-parameter fiberoptic sensor (Paratrend 7) can be used for continuous monitoring of hepatic microperfusion in a pig model of hepatic ischemia given endothelinA receptor antagonist (ETA-RA) or isotonic saline. Methods: Fourteen anesthetized swine were subjected to 2 h of hepatic vascular exclusion. The animals were randomized into two groups: control group (n = 7, saline solution iv) and therapy group (n = 7, ETA-RA). For evaluation of ischemia – reperfusion injury, the data of the multiple-parameter sensor ( pO2para, pCO2para, and pHpara) were compared with partial oxygen pressure in tissue ( ptiO2), laser Doppler flow, and systemic hemodynamic, metabolic data, and time course of transaminases. Results: In the control group 30 and 60 min after reperfusion, the following values were measured: ptiO2: 34.0 F 8.6/36.3 F 7.0 mm Hg ( P < 0.05 vs. preop.: 49.8 F 12.1 mm Hg), laser Doppler area: 133.3 F 23.2/156.4 F 15.4 ( P < 0.05 vs. preop.: 215.9 F 14.8). Animals in the therapy group revealed significantly improved values ( ptiO2: 54.0 F 8.6/58.1 F 7.8 mm Hg, laser Doppler: 210.2 F 38.5/225.2 F 21.3; P < 0.05). Using the Paratrend, also an improvement in the therapy group was seen 30 and 60 min after reperfusion. The values showed a strong correlation with ptiO2 (r = 0.895; P < 0.05) and laser Doppler flow (r = 0.807; P < 0.05). In the treatment group, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glutamate dehydrogenase (GLDH) were reduced 6 and 18 h after reperfusion, respectively, indicating hepatoprotection by the therapy ( P < 0.05 vs. control). Conclusions: The Paratrend sensor offers the opportunity to study postischemic organ hemodynamics through the simultaneous measurement of interstitial pH, pCO2, and pO2 in a small tissue region. This method offers a prognostic tool for the study of the effects of experimental vasoactive therapy on liver microcirculation and perspectives for continuous monitoring of human liver microperfusion after liver surgery and trauma. D 2003 Elsevier Inc. All rights reserved. Keywords: Ischemia – reperfusion; Liver; Hepatic

Introduction In hepatic ischemia – reperfusion, microcirculatory dysfunction has to be considered as a primary process that triggers the final manifestation of tissue injury. Postischemic microcirculatory dysfunction includes the failure of sinusoidal perfusion, that is, the no-reflow, which is caused by endothelial swelling, intravascular hemoconcentration, and * Corresponding author. 2nd Department of Surgery, University of Leipzig, Liebigstr. 20a, 04103 Leipzig, Germany. Fax: +49-341-97-17209. E-mail address: [email protected] (D. Uhlmann). 0026-2862/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2003.09.002

a dysbalance between the vasoactive mediators endothelin (ET) and nitric oxide (NO). Apart from perfusion failure, ischemia – reperfusion further promotes a microcirculationassociated inflammatory response (reflow paradox) that involves the release of aggressive mediators, such as oxygen radicals, tumor necrosis factor alpha and interleukin-1, the upregulation of leukocytic and endothelial adhesion molecules (selectins, beta-integrins, ICAM-1), and the interaction of platelets and leukocytes with the microvascular endothelium. The regional damage of microcirculation followed by a reduction in tissue oxygenation may be a major factor in the

D. Uhlmann et al. / Microvascular Research 67 (2004) 38–47

development of hepatic dysfunction. Significant organ injury may be prevented if hepatic microcirculatory dysfunction is detected early and therapeutic efforts are directed at reversing this regional hypoxia. Connett et al. (1990) suggested that the dysoxic threshold can be identified by measuring intracellular pO2, along with an indicator of cellular metabolism. Most clinical assessments are based on determination of whole body oxygen delivery and consumption, which provide limited information on hepatic dysoxia (Kruse, 1999). Hepatic dysoxia might be identified through oxygen delivery and consumption measurements from portal and hepatic blood, although this measurement is cumbersome and not continuous. Schlichtig and Bowles (1994) showed that intestinal dysoxia and anaerobic metabolic acidosis can be measured by using tissue pCO2 electrodes. Recently, fiberoptic sensors capable of measuring pCO2, pH, and pO2 have been shown to be useful for assessing hemorrhagic shock and resuscitation in the interstitium of skeletal muscle (McKinley et al., 1998a,b). These approximately 0.5-mm-diameter sensors, originally designed for intravascular blood gas measurement, can be inserted directly into tissue to measure pH and gas partial pressures of the interstitial fluid. pCO2, pH, and pO2 values determined in the interstitium of skeletal muscle have been shown to be more sensitive indicators of the degree of shock and adequacy of resuscitation than conventional systemic measures or gastric tonometric parameters (McKinley and Butler, 1999). This single small sensor provides the opportunity to simultaneously measure a parameter that approximates intracellular pO2, as suggested by Connett, along with two parameters that reflect anaerobic metabolism during conditions of reduced oxygen delivery (tissue pH and pCO2) in almost exactly the same region of tissue (McKinley and Butler, 1999). For such a sensor to be acceptable in the study of microcirculatory dysfunction and development of successful therapeutic strategies, its comparison with wellestablished methods of evaluation of microcirculation is needed. Therefore, the purposes of this study were (a) to determine whether several parameters that are altered during the process of hepatic ischemia – reperfusion could be assess by using a fiberoptic sensor, and (b) to evaluate this new method to predict the effect of a selective endothelinA receptor antagonist (ETA-RA) to improve ischemia – reperfusion-related microcirculatory disturbances. ET-1 is thought to be one of the mediators involved in the pathophysiology of microcirculatory disturbance induced by ischemia –reperfusion (Goto et al., 1994; Kawamura et al., 1995; Nakamura et al., 1995). Within the endothelin family, ET-1 is the most abundant and potent member. It was originally isolated from cultured porcine endothelial cells and was found to be a powerful vasoconstrictor (Yanagisawa et al., 1988). The actions of ET-1 which acts mainly as

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a paracrine factor are mediated by two receptor subtypes, designated ETA and ETB (Clozel et al., 1992; Yanagisawa et al., 1988). ETA receptors mediate vasoconstriction, ETB receptors primarily NO-dependent vasodilation, and ET degradation. Therefore, selective ETA receptor blockade may offer a successful means of hepatoprotection by the protection of postischemic microcirculation.

Materials and methods Experimental protocol All animals were treated in accordance with the ‘‘Guiding Principles in the Care and Use of Animals’’ approved by the Council of The American Physiological Society. The experiments conformed to the national guidelines for animal care and were approved by the Institutional Animal Care and Ethics Committee (Regierungspra¨sidium Leipzig, Germany; No. 02/00). Fourteen female German Landrace pigs weighting 20– 25 kg were used and randomized into two groups. The control group (n = 7) received 50 ml of isotonic saline solution iv, and the therapy group (n = 7) received the specific ETA-RA BSF 208075 (5 mg/kg iv in 50 ml saline) provided by Knoll AG, Ludwigshafen, Germany. The ETARA was administered as continuous intravenous infusion during the first 20 min of reperfusion. The animals were humanely euthanized after a 7-day observation period. Anesthesia and surgical procedure Before induction of anesthesia, a premedication with a dose of 4 mg/kg of azaperon (StresnilR, Cilag-Janssen, Neuss, Germany), 2 mg/kg of ketanest (KetaminR, Ratiopharm, Ulm, Germany), and 0.2 mg/kg of atropine sulfate (AtropinsulfatR, Braun, Melsungen, Germany) was administered intramuscularly. An ear vein was punctured, an intravenous line was placed, and anesthesia was induced with a bolus dose of 5 mg thiopenthal (TrapanalR, Byk Gulden, Konstanz, Germany). After the trachea was intubated, the animals were placed in a dorsal recumbent position. A Draeger ‘‘Julian’’ ventilator (Draegerwerk AG, Luebeck, Germany) was connected, and the pigs were ventilated with an oxygen –air mixture with a tidal volume of approximately 10 ml/kg at an appropriate respiratory rate of 16/min to produce normocapnia (PaCO2 36 –43 mm Hg) at baseline conditions. FIO2 was set at 0.4 to assure good oxygenation (PaO2 100 – 200 mm Hg) throughout the experiment. A positive end-expiratory pressure of 4 cm H2O was used. The anesthesia was maintained with 0.75 – 1.5% isoflurane (ForeneR, Abbott, Wiesbaden, Germany) and fentanyl (FentanylR Janssen, Neuss, Germany, 0.05 mg/h iv). Isotonic saline [10 ml/(kg/h)] was given for basal fluid requirements and replacement of perioperative losses. Normothermic temperatures (38 F 0.5jC) were maintained with the use of

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prewarmed intravenous fluids and a thermostatically controlled heating pad. The mean blood loss during the whole experiment was 202 F 66 ml, (i.e., less than 10% of the circulating blood volume). For substitution, the animals received 250 to 400 ml of Gelatine (GelafusalR M, SerumWerk Bernburg, Germany). According to the blood gas analyses, the pigs were given sodium bicarbonate (1 mol, 8.4%, Serag-Wiessner, Naila, Germany). An arterial line was inserted into the right carotid artery for recording of arterial pressure. Blood pressure was recorded with Ohmeda transducers (Medical Devices Division, USA) connected to a Siemens Monitor 1281 (Siemens Medical Electronics, USA). After midline incision, the hepatoduodenal ligament and the subhepatic and suprahepatic caval vein were prepared and cross-clamped for 120 min. To avoid portal consumption and to maintain macrohemodynamics, a porto- or femorojugular shunt was inserted. Paratrend After laparotomy, a multiparameter pO2para, pCO2para, and pHpara sensor was inserted into the parenchyma of the left liver lobe. There was a distance of about 3 cm between Licox and Paratrend sensors. The sensor used in this study has been designed for intraarterial on-line blood gas monitoring and contains two modified optical fibers: for the measurements of pO2, pCO2, and pH, a miniaturized polarographic Clark oxygen electrode, and a thermocouple for temperature measurement (Paratrend 7, Biomedical Sensors Ltd, High Wycombe, UK). The void between each sensor is filled with acrylamide gel containing phenol red. The cylindrical construction of the sensor allows measurement over the entire surface of the probe. The pH and pCO2 sensors work on the principle of optical absorption. However, the pO2 sensor works on the principle of fluorescence quenching, whereby the intensity of a fluorescent optical emission from an indicator is reduced in the presence of oxygen. The diameter of the probe is 0.5 mm and the four sensing components are located at different intervals along the distal 4 cm of the polyethylene probe permeable for O2 and CO2. The outer surface of the probe is coated with covalently bonded heparin to prevent fibrin deposition. The flexible wire part of the device has a length of 600 mm. The monitor continuously displays pH, pCO2, pO2, and temperature. The readings are given at the actual temperature of the animal. The Paratrend sensors were calibrated before use in a separate calibration unit (Diametrics Medical) by using three different calibration gases (CO2/oxygen/N2 = 2:0:98, 7:15:78, and 22.5:50:27.5). During the experimental protocol, measurements for each parameter were made every 10 s and recorded on a laptop computer. Partial oxygen tension in the tissue Continuous measurement of the partial oxygen tension in the liver tissue ( ptiO2) was performed by insertion a Clarke-

type electrode (Licox, Gesellschaft fu¨r medizinische Sondentechnik mbH, Kiel-Mielkendorf, Germany) into the parenchyma of the left liver lobe. After an equilibration time, tissue ptiO2 was measured after laparotomy for 30 min, during the whole time of ischemia (120 min), and for 120 min after reperfusion. Laser Doppler flow measurement Blood flow was measured by placing the Doppler flow probe at the left liver lobe. The measurements were monitored on a Moor Instruments DRT4 Monitor (Moor Instruments, Devon, UK). The principle of the probe is that light generated by a laser diode (780-nm wavelength with maximal emission energy of 1.0 mW) penetrates the tissue where it is reflected by circulating blood cells. Analog laser Doppler flow signals were digitalized and processed on a personal computer with the DRTSOFT V2.9 software provided (Moor Instruments, Devon, UK). Blood flow was recorded for at least 30 s after a stable signal was obtained. Data are given as integral under the curve. For integral estimation, the mean of the pulse waves within the 30-s sampling period was calculated. Portal venous and hepatic arterial flow Portal venous and hepatic arterial flow were continuously monitored by an ultrasonic transit time flowmeter (Model T201, Transonic Systems, Ithaca, USA). Blood gas Blood gas samples were examined in the automated blood gas analyzer (ABL 300; Radiometer, Copenhagen, Denmark) and with the oximeter calibrated for pig hemoglobin. Serum levels of aspartate aminotransferase, alanine aminotransferase, and glutamate dehydrogenase (GLDH) Blood samples were drawn from central venous blood. The probes were centrifuged for 15 min at 4000 rpm and 4jC. The serum aliquots were stored at 80jC. Serum aspartate aminotransferase (ALT), alanine aminotransferase (AST), and glutamate dehydrogenase (GLDH) were measured on an automatic analyzer (Hitachi 917) by photometry (Kraft and Du¨rr, 1999). Histology Specimens were taken from the left lobe of the liver, fixed in 4% formaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin –eosin. Histomorphological alterations were semiquantitatively assessed by a scoring system from absent to severe. Impairment before organ manipulation was assessed by four parameters, injury during warm ischemia, reperfusion and follow up was assessed by 10

D. Uhlmann et al. / Microvascular Research 67 (2004) 38–47 Table 1 Semiquantitative histological score

Before manipulation Activation of Kupffer cells Portal infiltration Steatosis Intralobular necrosis

Slight

Moderate

Severe

1

1

1

0 1 3 (single cell-)

1 3 8 (group cell-)

2 5 15 (mass cell-)

4 4 4

6 6 6

3 0

5 2

8 (group cell-) 4 (group cell-) 0

15 (mass cell-) 8 (mass cell-) 1

1 (subcapsular)

3 (interstitial)

8

15

Warm ischemia, reperfusion, follow up Interstitial edema 2 Intracellular swelling 2 Quality of sinusoidal 2 space Steatosis 1 Reaction of Kupffer 0 cells Intralobular necrosis 3 (single cell-) Subcapsular necrosis 1 (single cell-) Reaction of liver 0 capsule Hemorrhage 0 Only after reperfusion Hyperaemia 2

parameters (Table 1). Score points were assigned according to the importance of each parameters for organ function. Score values were given as percentage of maximal attainable score points. Statistical analysis Data were expressed as means F standard deviation (SD). Variable were tested for group differences with the Mann –Whitney U test. Correlation between the methods was analyzed by the Spearman rank correlation test. The

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degree of agreement between Paratrend and Licox sensors was assessed according to Bland and Altman (1986). P values < 0.05 were considered significant.

Results Macrohemodynamic and blood gas changes There were no differences in mean arterial pressure in the control and therapy groups before surgery (72.4 F 11.1 vs. 71.3 F 9.7 mm Hg). The values dropped during clamping to 63.2 F 8.4 and 63.1 F 9.4 mm Hg after 120 min of vascular exclusion. After reperfusion the arterial pressure showed no raise and did not differ significantly between both investigated groups, despite the vasoactive drug use in the therapy group (Tables 2 and 3). The changes in arterial blood gases are also given in Tables 2 and 3. There were no significant changes between the groups in all measured parameters. Portal venous and hepatic arterial flow Portal venous flow in the control and therapy groups before surgery were 394.1 F 42.1 and 388.5 F 39.5 ml/ min. Thirty minutes after reperfusion, values of 349.0 F 32.8 and 402.3 F 34.1 ml/min, and 60 min after reperfusion, 352.2 F 23.5 and 389.4 F 34.2 ml/min were measured, respectively. Hepatic arterial flow at baseline was 129.5 F 8.9 ml/min in the control group and 122.4 F 10.2 ml/min in the therapy group. Thirty minutes after reperfusion, a decrease to 67.2 F 11.2 (control group, P < 0.05) and 104.6 F 32.1 ml/min (therapy group) was found. Sixty minutes after reperfusion,

Table 2 Hemodynamic, metabolic, and hepatic microcirculatory data in the control group (mean F SD) Parameter

Baseline

Heart rate (beats/min) Mean arterial pressure (mm Hg) Central venous pressure (mm Hg) a-Base excess a-pH a-pCO2 (mm Hg) a-pO2 (mm Hg) Hemoglobin (g/dl) Hematocrit (%) Leukocytes (103/mm3) Platelets (103/mm3) Arterial oxygen saturation (%) Paratrend pH Paratrend pCO2 (mm Hg) Paratrend pO2 (mm Hg) Licox ptiO2 (mm Hg) Laser Doppler

90.0 72.4 9.2 1.4 7.45 41.4 169.9 8.0 24.5 17.7 457.6 69.8 7.38 53.2 54.2 49.8 215.9

F F F F F F F F F F F F F F F F F

Ischemia (120 min) 12.1 11.1 2.6 1.1 0.08 5.3 21.8 1.3 4.1 3.3 218.6 13.7 0.18 8.2 12.2 12.1 14.8

112.0 63.2 9.2 2.5 7.34 33.8 208.3 7.2 22.3 16.1 468.9 74.2 6.75 165.6 9.5 2.5 17.2

F F F F F F F F F F F F F F F F F

Reperfusion 30 min 23.6 85.4 2.7 2.7 0.05 4.5 17.3 1.2 3.9 8.2 196.8 10.2 0.15 15.1 4.0 0.8 2.3

114.0 63.0 9.6 2.2 7.34 45.1 175.9 8.8 27.4 12.2 424.4 88.3 7.25 70.9 37.0 34.0 133.3

F F F F F F F F F F F F F F F F F

60 min 19.2 8.3 1.8 1.5 0.01 6.8 9.0 3.7 12.2 8.1 194.9 7.6 0.23 5.2 8.5 8.6 23.2

109.0 61.6 9.6 1.4 7.41 36.1 178.9 7.2 21.9 15.4 422.6 88.0 7.31 65.7 39.4 36.3 156.4

F F F F F F F F F F F F F F F F F

9.6 7.1 1.8 2.4 0.03 3.0 16.5 1.6 5.4 6.8 176.9 9.6 0.12 5.5 3.5 7.0 15.4

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65.5 F 21.3 ml/min in the control group and 97.4 F 17.7 ml/min in the therapy group were measured. Microhemodynamic changes Partial oxygen tension in the tissue At baseline, in both groups, physiologic tissue oxygenation with values of 49.8 F 12.1 mm Hg (control group) and 48.7 F 8.6 mm Hg (therapy group) were found measured with the Licox device. After 120 min of normothermic ischemia, values dropped to 2.5 F 0.8 mm Hg (control group) and 2.7 F 0.7 mm Hg (therapy group). After reperfusion, the changes in tissue oxygenation occurred in two phases. Firstly, 10 min after reperfusion, there was a significant increase in ptiO2. After 20– 30 min of reperfusion, the ptiO2 then reduced to a plateau of 34.0 F 8.6 mm Hg at 30 min and 36.3 F 7.0 mm Hg 60 min after reperfusion, but failed to restore preischemic values. Treatment: ETA-RA treatment did not change values during ischemia compared to the control group, but led to fast restoration of tissue ptiO2 to 54.0 F 8.6 mm Hg after 30 min and to 58.1 F 7.8 mm Hg after 60 min of reperfusion ( P < 0.05 vs. control). The postischemic oxygenation in the therapy group showed values comparable to preischemic values. Laser Doppler flowmetry In both groups, the laser Doppler values displayed as integrals under the curve revealed no significant differences between the control (216.6 F 14.8) and therapy group (222.5 F 16.8). One hundred twenty minutes of normothermic ischemia resulted in a decrease in erythrocyte flux to an integral of 19.8 F 5.7 and 22.3 F 6.8. Thirty and sixty minutes after reperfusion, a significant decrease of blood flow compared to the preischemic levels was found in the control group (133.3 F 23.4/ 156.4 F 15.4). Treatment: ETA-RA therapy averted the decrease in erythrocyte flux. Thirty and sixty minutes after reperfusion, integrals of 212.0 F 12.6 and 219.8 F 6.9 were seen ( P < 0.05 vs. control group). These values were comparable to preischemic measurements. Aspartate aminotransferase, alanine aminotransferase, and glutamate dehydrogenase Serum AST, ALT, and GLDH as markers of hepatic injury were within the normal range in the control and therapy groups before surgery (AST: 0.47 F 0.07 and 0.44 F 0.10 Amol/l; ALT: 0.50 F 0.10 and 0.47 F 0.16 Amol/l; GLDH: 0.02 F 0.01 and 0.03 F 0.01 Amol/l). Eighteen hours after reperfusion, AST (27.42 F 3.92 Amol/l) and ALT (2.2 F 0.35 Amol/l), and 6 h after reperfusion, GLDH (0.70 F 0.09 Amol/l) activities showed peak values and were significantly increased compared to basal levels ( P < 0.05), reflecting the substantial loss of hepatocellular integrity (Fig. 1).

Fig. 1. Serum levels of aspartate aminotransferase before ischemia, at the end of ischemia, and from 30 min to 7 days after reperfusion given endothelinA receptor antagonist (therapy group) or saline (control group). The serum levels were significantly lower 2 h to 3 days after reperfusion in the therapy group as compared to the control group. *P < 0.05 compared with control group. Values are means F SD.

Treatment: In the treatment group, AST (17.20 F 3.77 Amol/l; P < 0.05 vs. control) and ALT (1.65 F 0.36 Amol/l; P < 0.05 vs. control) 18 h after reperfusion were significantly lower in the therapy group compare to control animals. GLDH peak 6 h after reperfusion (0.38 F 0.08 Amol/l; P < 0.05 vs. control) was significantly reduced. Histology Before liver manipulation, there was no evidence of relevant morphological damage in either group. At the end of the warm ischemic time, a slight increase of injury was seen in the control and therapy groups (interstitial and intracellular edema, single necrotic cells, and activation of Kupffer cells) without difference between the two groups (control group: 19%, therapy group: 16% injury). Ten min after reperfusion, the histological injury increased in both groups, but was found to be significantly lower in the therapy group ( P < 0.05). A slight increase of edematous injury (approximately 36%), reaction of the capsule of the liver (86%), and of the Kupffer cells (71%) was discovered in the therapy group, whereas the histomorphological alterations in the control group included a strongly developed interstitial and intracellular edema (57%, 90%), irregular trabecular disruption, hemorrhage, invasion of inflammatory cells, dilatation of the sinusoidal space, and sinusoidal congestion. Summarized injury in the control group was 55% vs. 19% in therapy group ( P < 0.05). One and two hours after reperfusion, a further increase of morphological damage was found in control group (strong edema, hemorrhagic and inflammatory infiltration of the sinusoidal space, and increase of subcapsular and intralobular necrosis), whereas in the therapy group no relevant change of injury was found. Summarized, injury 2 h after

D. Uhlmann et al. / Microvascular Research 67 (2004) 38–47

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Table 3 Hemodynamic, metabolic, and hepatic microcirculatory data in the therapy group (mean F SD) Parameter

Baseline

Heart rate (beats/min) Mean arterial pressure (mm Hg) Central venous pressure (mm Hg) a-Base excess a-pH a-pCO2 (mm Hg) a-pO2 (mm Hg) Hemoglobin (g/dl) Hematocrit (%) Leukocytes (103/mm3) Platelets (103/mm3) Arterial oxygen saturation (%) Paratrend pH Paratrend pCO2 (mm Hg) Paratrend pO2 (mm Hg) Licox ptiO2 (mm Hg) Laser Doppler

88.7 71.3 9.1 1.7 7.41 42.1 166.2 8.2 25.1 16.6 451.3 72.7 7.32 52.1 53.4 48.7 220.5

F F F F F F F F F F F F F F F F F

Ischemia (120 min) 4.6 9.7 3.2 1.3 0.07 5.26 17.3 1.2 3.6 4.0 147.5 12.9 0.21 6.2 8.3 8.6 17.6

109.3 63.1 10.3 2.4 7.37 36.8 215.0 7.9 24.2 15.0 399.6 75.2 6.77 172.5 9.8 2.7 15.3

F F F F F F F F F F F F F F F F F

Reperfusion 30 min 21.8 9.4 3.7 2.9 0.11 7.52 21.8 1.6 4.7 4.3 136.2 15.0 0.13 12.6 3.2 0.7 3.5

103.6 59.7 10.0 5.1 7.26 39.8 182.0 6.9 20.8 14.2 376.4 84.0 7.30 56.3 59.2 54.0 210.2

F F F F F F F F F F F F F F F F F

60 min 16.5 9.8 3.6 2.1 0.09* 8.3 19.5 1.2 3.8 7.8 119.0 6.4 0.19 5.2* 8.5* 8.6* 18.5*

112.1 58.4 10.0 3.9 7.27 42.1 188.7 6.3 19.8 13.9 364.9 85.4 7.36 58.7 62.3 58.1 225.2

F F F F F F F F F F F F F F F F F

18.4 3.6 3.6 2.0 0.08* 10.5 6.8 1.1 3.6 6.4 129.6 8.0 0.11 6.5* 5.2* 7.8* 21.3*

* P < 0.05 compared with control group.

reperfusion in the control group was 60% vs. 15% in therapy group ( P < 0.05). During follow up (4 and 7 days postoperatively), an evident decrease of morphologic –pathologic alterations was observed in both groups in all investigated parameters. However, a significant difference in score values was still observed between the two groups (7 days postoperatively: control group 33%, therapy group 13%; P < 0.05). Paratrend Intrahepatic pCO2para, pO2para, and pHpara measurements at various stages of ischemia and reperfusion are shown in

Fig. 2. Partial oxygen pressure measured by Paratrend ( pO2para) in the liver before ischemia and from the beginning of reperfusion to 120 min after reperfusion, given endothelinA receptor antagonist (therapy group) or saline (control group). pO2para was significantly higher at each measurement point in the therapy group as compared to the control group. *P < 0.05 compared with control group. Values are means F SD.

Tables 2 and 3. Highly significant changes after the clamping ( P < 0.05 vs. baseline) were seen in hepatic oxygenation and the metabolic parameters registered by Paratrend. Mean pO2para decreased from baseline 54.2 F 12.2 mm Hg within few minutes and reached levels of about 10 mm Hg 120 min after vascular exclusion. In early reperfusion period, a biphasic course of pO2para values with an early peak at 10 min after reperfusion was found (Fig. 2). Treatment: According to laser Doppler measurement and Licox ptiO2 measurement, there were significant better values found in the therapy group compared to control. Thirty minutes after reperfusion, the pO2para showed a restoration of preischemic values ( P < 0.05 vs. control group). A significant improvement of liver oxygenation was

Fig. 3. The relationship between the changes in hepatic blood flow measured by laser Doppler flowmetry and partial oxygen pressure measured by Paratrend ( pO2para) 60 min after reperfusion. There is a significant correlation between both methods.

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Fig. 4. The relationship between the changes in partial oxygen pressure measured by Licox ( ptiO2) and partial oxygen pressure measured by Paratrend ( pO2para) 60 min after reperfusion. There is a significant correlation between both methods.

Fig. 6. The relationship between the changes in peak GLDH values 6 h after reperfusion and partial oxygen pressure measured by Paratrend ( pO2para) 60 min after reperfusion. There is a significant correlation between the two parameters.

also measured in the therapy group 60 min after reperfusion ( P < 0.05 vs. control group). pCO2para and pHpara changed gradually during the whole clamping period. Mean pCO2para (baseline: 53 mm Hg) increased to about 170 mm Hg 120 min after clamping. Intrahepatic pCO2para values in the therapy group were significantly lower 30 and 60 min after reperfusion as compared to the control group ( P < 0.05). Intrahepatic pHpara (baseline: 7.38) reached mean values of 6.75 just before declamping. The postischemic intrahepatic pHpara was higher in the therapy group, however, without significance.

Correlation between Paratrend pO2 and pO2 Licox after reperfusion Thirty minutes (r = 0.853, P < 0.05) and 60 min (r = 0.895, P < 0.05) after reperfusion, a significant correlation between pO2para and ptiO2 was seen (Fig. 4). A high degree of agreement between pO2para and ptiO2 is documented in Fig. 5 according to Bland and Altman (1986).

Correlation between Paratrend pO2 and laser Doppler flowmetry after reperfusion The pO2para correlated with laser Doppler flowmetry 30 min (r = 0.752, P < 0.05) and 60 min (r = 0.807, P < 0.05) after reperfusion (Fig. 3).

Fig. 5. Good agreement between pO2para and ptiO2 in the quantification of partial oxygen pressure using the Bland and Altman analysis.

Correlation between Paratrend pO2 and GLDH after reperfusion For correlation, peak GLDH values 6 h after reperfusion and pO2para measurements 60 min after reperfusion were used. A significant correlation between pO2 and AST was seen (r = 0.869, P < 0.05; Fig. 6).

Discussion Liver ischemia remains one of the most important problems associated with hepatobiliary surgery. Portal triad clamping is a common and simple procedure for reducing operative bleeding from the raw surface during hepatic parenchymal transection (Delva et al., 1989; Sano et al., 1999). However, it does not prevent reflux bleeding through branch hepatic veins. Total hepatic vascular exclusion consists of portal triad clamping and occlusion of the inferior vena cava below and above the liver. This procedure is often used for major resections of large and posterior liver tumors to avoid massive hemorrhage and air embolism caused by tearing of the retrohepatic vena cava or hepatic veins (Bismuth et al., 1989; Delva et al., 1984). Clinical and experimental studies have shown that the hepatic tolerance to warm ischemia may last approximately 90 min in the human being (McKeown et al., 1988) and 120 min in the pig. To induce a severe ischemic trauma, we used a pig model of 120-min total vascular

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exclusion of the liver and portal decompression by a portosystemic shunt (Nordlinger et al., 1980). Numerous methods have been applied to quantify hepatic microvessel perfusion, like indocyanine green clearance (Wang et al., 1991), indicator dilution (Huet et al., 1973), fractionation techniques (Sapirstein, 1967), electromagnetic (Hopkinson and Schenk, 1968) and Doppler flow probes (Loisance et al., 1973), inert gas clearance (Leiberman et al., 1978; Mathie, 1986), and laser Doppler flowmetry (Almond and Wheatley, 1992). All of these methods mentioned above preclude the continuous monitoring of hepatic perfusion in patients over several days after liver surgery or transplantation. The multiple-parameter fiberoptic sensors have been proven to be reliable for continuous microcirculatory assessment in this study of ischemia-induced hepatic injury. They permit tissue-acid base assessment in solid organs. The small size makes it possible to consider clinical applications because its insertion in skeletal muscle or solid organs should carry minimal risk. McKinley et al. (1998a,b) used a similar fiberoptic sensor to demonstrate trends in muscle pH, pCO2, and pO2 for a trauma patient admitted with severe hemorrhagic shock. These measurements in the muscle of a human subject demonstrate similar trends to those observed in swine studies (Puyana et al., 2000). Venkatesh et al. (2000) used multiple-parameter fiberoptic sensors to simultaneously measure subcutaneous pO2 and ileal pCO2 in rats subjected to hemorrhagic shock. There are also experiences for the use of Paratrend in the brain and spinal cord (Hellberg et al., 2001; Hutchinson et al., 2000). The Paratrend sensors permit the opportunity to collect data every 10 s, providing a larger number of data points than other intermittent measurements that have been used previously to assess ischemia. Previous work by Walley et al. (1998) suggests that any organ can be divided into a collection of tissue elements. Each of these tissue elements will have a range of oxygen delivery and oxygen consumption values. This theoretical model yields a curvilinear relationship between oxygen delivery and oxygen consumption that is a function of the dispersion, or the degree of heterogeneity, in oxygen delivery and consumption in the tissue elements. Although our sensor is small, the active area of the oxygen-sensing element is estimated to be 2.75 mm2, whereas the pCO2 and pH sensors have an approximate surface area of 1.8 mm2. Within that region of tissue, it is likely that there is heterogeneity in pO2 and oxygen consumption. The physiology and time course of ischemia-induced flow and PO2 reduction are complex. As ischemia starts, pCO2 accumulates, and because pCO2 is in equilibrium with HCO3 , increasing pCO2 results in increasing H+ or declining pH. When ischemia continues to bring pO2 to the critical value, dysoxia commences and further increases in pCO2 and H+ are the result of anaerobic metabolism (Knichwitz et al., 1998; Schlichtig and Bowles, 1994).

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Soller et al. (2001) first reported critical values for hepatic tissue pCO2 (64 – 76 Torr) and pH (7.19 – 7.29), and they are comparable to critical values determined for hollow organs such as the stomach and intestine by using tonometers. By using tonometers, Schlichtig and Bowles (1994) first suggested that the critical pCO2 ( pCO2crit) in the canine intestine is between 65 and 75 Torr (8.7 – 10.0 kPa). Walley et al. (1998), in a swine model of hemorrhagic shock, used tonometer data to estimate a gastric pCO2crit of 103 F 40 Torr (13.7 F 5.3 kPa) and a small bowel pCO2crit of 57 F 15 Torr (7.6 F 2.0 kPa). Critical pH values from these reports were estimated as 7.04 F 0.14 (stomach) and 7.29 F 0.15 (small bowel). In later work, Schlichtig and Bowles (1994) used pCO2 electrodes to assess pCO2crit on the mucosal and serosal surfaces of swine intestines during flow reduction. Mucosal pCO2crit was found to be 129 F 34 Torr (17.2 F 4.5 kPa), whereas serosal pCO2crit was 96 F 21 Torr (12.8 F 2.8 kPa). These authors suggested that tonometric measurements underestimate pCO2 critical values because the tonometers occupy much of the intestinal lumen and may deplete tissue pCO2. Their data indicate that there will be organ-specific values for pCO2crit and pHcrit, and that heterogeneity of blood flow (or cellular metabolism) within an organ can lead to different values at different locations within the same organ. While pCO2 in our study in the control group 30 min after reperfusion is in the critical range (70.9 F 5.7 Torr), pH values showed a good stabilization after reperfusion. In the therapy group, critical values of pCO2 (56.3 F 5.2 and 58.7 F 6.5 mm Hg) and pH (7.30 F 0.19 and 7.36 F 0.11) were not seen over the whole observation period. Our study may have been impacted by two limitations of the multiple-parameter fiberoptic sensor. The maximum measurable pCO2para is 199 Torr (26.5 kPa). This limitation may have introduced small errors in the critical values determined for pCO2. Because oxygen delivery in the liver can be heterogeneous, measurements made in one part of the liver may not indicate dysoxia in another part of the liver. A better approach would use several sensors at various locations in the liver. The small size of the probe, coupled with the high sensitivity of the oxygen sensor, makes the sensor very susceptible to high tissue pO2 readings when the sensor is positioned near an artery. A further advantage of the Paratrend is that it is available in most surgical departments on the intensive care unit. To evaluate this new method of microcirculatory assessment, we investigated the effect of a selective ETA receptor antagonist to improve hepatic ischemia –reperfusion-related microcirculatory disturbances. Vasoconstriction is a relevant factor involved in microcirculatory disturbance during organ reperfusion. ET-1 is one of the most potent endotheliumderived constrictor agents (Goto et al., 1994; Kawamura et al., 1995; Nakamura et al., 1995; Yanagisawa and Masaki, 1989). It has been demonstrated that hepatic nonparenchymal cells as well as vascular endothelial cells produce ET-1

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(Rieder et al., 1991). ET-1 is thought to potentiate hepatic ischemia –reperfusion injury, but the mechanism involved has not been fully studied under these conditions. In the current study, we have found evidence that the blockade of ETA receptors during reperfusion after warm ischemia led to improved hepatic microperfusion. This beneficial effect was characterized by a significant improvement of laser Doppler flow and local oxygen pressure and a reduction of pCO2para in the therapy group. In accordance with El-Desoky et al. (2001), we found an increase of ptiO2 and pO2para in the control and therapy groups within the first minutes after reperfusion, rapidly followed by capillary perfusion failure. Similar observations were made by Kubes et al. (1997) in the intestine. Based on the present study using continuous tissue oxymetry, the ‘‘reflow paradox’’ described by Menger et al. (1992) seems to be the consequence of a short reperfusion period. During this short period of capillary perfusion after prolonged ischemia, the whole cascade of reperfusion events, such as formation of oxygen free radicals and release of proinflammatory mediators, is induced. However, as discussed above, it is a considerable benefit that during the transient capillary reperfusion after unclamping of the supply vessels there is a window for prophylactic treatment of microcirculation which we used in our study design. Compared with the control group, prophylactic application of the selective ETA-RA led to significantly higher pO2paravalues throughout the 7-day period of measurement and to levels that did not differ from the preischemic capillary perfusion. Likewise, a significantly better blood flow assessed by laser Doppler flowmetry was seen in the therapy group early after reperfusion. To detects differences in liver microhemodynamics, laser Doppler (Almond and Wheatley, 1992) and Licox (Hiratsuka et al., 2000; Mucke et al., 2000) have been proven. We could show that the Paratrend is equally useful for detection of microcirculatory changes. Further on, the Paratrend, if it remains in situ, can detect microcirculatory disturbances some hours and days after surgery, that is, on the ICU and gives additional information about the metabolic state of the organ, by analyzing pCO2, pH, and temperature. A further advantage of the Paratrend is that it is available on the intensive care unit in most surgical departments. We have shown that this multiple-parameter fiberoptic sensor designed for intravascular blood gas and pH measurement can be used to simultaneously assess pH, pCO2, and pO2 of the interstitial fluid of the liver parenchyma and that these values can be used to assess liver microperfusion. Thus, an early intervention is possible if severe damages of microcirculation are seen. By this way, outcome of liver surgery and liver transplantation could be improved. In another study, we could show the rapid detection of occlusion of hepatic artery or portal vein after liver surgery and the possibility to differentiate between occlusion of one of the vessels (Uhlmann et al., 2002).

Acknowledgments This study was supported in part by a grant from the Else Kro¨ner-Fresenius Stiftung and by a junior research fund of the Medical Faculty at the University of Leipzig. We thank Christoph Peuse (Diametrics Medical Ltd.) for providing the Paratrend sensor and the team of the Medical Experimental Centre at the University of Leipzig for professional assistance.

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