Heparin and the nonanticoagulant N-acetyl heparin attenuate capillary no-reflow after normothermic ischemia of the lung

Heparin and the nonanticoagulant N-acetyl heparin attenuate capillary no-reflow after normothermic ischemia of the lung

Heparin and the Nonanticoagulant N-Acetyl Heparin Attenuate Capillary No-Reflow After Normothermic Ischemia of the Lung Takayuki Nakamura, MD, Brigitt...

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Heparin and the Nonanticoagulant N-Acetyl Heparin Attenuate Capillary No-Reflow After Normothermic Ischemia of the Lung Takayuki Nakamura, MD, Brigitte Vollmar, MD, Johannes Winning, MD, Mitsuomi Ueda, MD, Michael D. Menger, MD, and Hans-Joachim Scha¨fers, MD Department of Thoracic and Cardiovascular Surgery and Institute for Clinical and Experimental Surgery, University of Saarland, Homburg/Saar, Germany, and Department of Thoracic Surgery, Kyoto University, Kyoto, Japan

Background. Ischemia-reperfusion injury of the lung frequently occurs after cardiopulmonary bypass, after pulmonary thromboendarterectomy, and especially after lung transplantation. Heparin is known to be protective in ischemia-reperfusion injury, but the risk for bleeding disorders may restrict its use in a variety of diseased conditions. Therefore, we tested the efficiency of nonanticoagulant N-acetyl (NA) heparin to protect from postischemic reperfusion injury of the lung. Methods. Pentobarbital-anesthetized, mechanically ventilated Lewis rats were heparinized (100 IU/kg) before insertion of catheters. Additionally, animals received either heparin (200 IU/kg; n ⴝ 7), NA heparin (1.1 mg/kg; n ⴝ 7), or saline (control, n ⴝ 7) before ischemia. After normothermic ischemia for 50 minutes, the left lung was reperfused for 120 minutes, or until the death of the animal. The nonischemic right lung was excluded after 10 minutes of reperfusion. Results. Survival rate at 120 minutes of reperfusion was 7 of 7 and 6 of 7 in the heparin and the NA-heparin group, but 0 in 7 in the control group (p < 0.01). At 30 minutes of reperfusion, PaO2, blood flow through the

ascending aorta and mean systemic blood pressure were also significantly higher in the heparin and the NAheparin group when compared with the control group (p < 0.05). Pulmonary vascular resistance was significantly lower in the heparin and the NA-heparin groups, and histologic examination of the lungs from these groups confirmed reperfusion of nutritive alveolar capillaries by the presence of red blood cells. Lack of red blood cells in the alveolar capillaries of lung specimens from the control group indicated failure of capillary reperfusion. Conclusions. Heparin and NA heparin exert similar protection against capillary no-reflow after normothermic ischemia of the lung. This implies that the protective effect of heparin is mediated by properties different from its anticoagulant activity. Thus the nonanticoagulant N-acetyl heparin may pose a safe new therapeutic approach in lung ischemia-reperfusion injury without increasing the risk of hemorrhagic complications.

P

dependent vascular relaxation following I/R by mechanisms independent of its anticoagulant properties [4]. Using an isolated lung perfusion model of the rabbit, it was further shown that selective O-desulfation of heparin inhibits complement-associated lysis of red blood cells, and thereby reduces I/R-associated pulmonary weight gain [5]. With these sustained pharmacological capabilities, modified heparins might offer therapeutic potential aiming to counteract I/R injury of the lung. Therefore, the purpose of the present study was to determine if the administration of heparin and the nonanticoagulant N-acetyl (NA) heparin could protect the in vivo rat lung from irreversible injury from a period of warm I/R.

revention or limitation of reperfusion injury is crucial for success of operative procedures including pulmonary ischemia, such as cardiopulmonary bypass, pulmonary thromboendarterectomy, and lung transplantation. Although the primary therapeutic use of heparin relates to its anticoagulant activity, this compound has been reported to protect skeletal and cardiac muscle subjected to ischemia-reperfusion (I/R) [1, 2]. Moreover, heparinization before ischemia has been demonstrated to exhibit a variety of beneficial effects on postischemic cell and organ function, which may not be attributed to its accelerating effect on antithrombin-proteinase reactions associated with its well-known ability to inhibit hemostatic mechanisms. Sternbergh and coworkers [3] reported that heparin prevents postischemic endothelial dysfunction and improved the depressed endothelium-

(Ann Thorac Surg 2001;72:1183–9) © 2001 by The Society of Thoracic Surgeons

Material and Methods

Presented at the Thirty-seventh Annual Meeting of The Society of Thoracic Surgeons, New Orleans, LA, Jan 29 –31, 2001.

Animals

Address reprint requests to Dr Vollmar, Institute for Clinical and Experimental Surgery, University of Saarland, 66421 Homburg/Saar, Germany; e-mail: [email protected]

Male Lewis rats weighing about 300 g were purchased from Charles River Laboratories (Sulzfeld, Germany). All animals were kept according to the “Guide for the Care

© 2001 by The Society of Thoracic Surgeons Published by Elsevier Science Inc

0003-4975/01/$20.00 PII S0003-4975(01)02959-9

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and Use of Laboratory Animals” (National Institutes of Health Guide, Vol. 25, No. 28; 1996) and experiments were approved by the local governmental institution.

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ations, whereas the dosage of 300 IU/kg heparin is used in cardiopulmonary bypass procedures.

Hemodynamic Measurements Anesthesia and Operation After intraperitoneal anesthesia with sodium pentobarbital (50 mg/kg, intraperitoneal), the animals were given tracheotomies and mechanically ventilated with room air using a volume-controlled respirator (Rodent Ventilator 683, Harvard apparatus, South Natick, MA). At a ventilatory rate of 40 minutes⫺1, tidal volume was set at 1.0 mL/100 g body weight (bw), and positive end-expiratory pressure was kept at 2 cmH2O. Animals were placed in supine position on a heating pad, maintaining body temperature at 36°C to 37°C. A left common carotid arterial catheter (PE-50, inner diameter 0.58 mm, Portex, Lythe, UK) was inserted to monitor systemic blood pressure and to obtain arterial blood for gas analysis. The arterial catheter further served for continuous infusion of isotonic saline solution (3 mL/hour) and for application of drugs. After opening of the thorax by a bilateral transverse thoracotomy in the fourth intercostal space, the right pulmonary artery, the right main bronchus, the left pulmonary artery, and the left main bronchus were isolated by means of a stereomicroscope. After bolus injection of heparin (100 IU/kg intraarterial), a 24Gcatheter (Neoflon, Ohmeda, Helsingborg, Sweden) was inserted into the pulmonary arterial trunk, fixed with a purse string suture (8-0 thread; Prolene, Ethicon, Germany), and continuously flushed by isotonic saline solution (2 mL/hour). An ultrasonic flow probe (2SB 546, Transonic Systems, Ithaca, NY) was placed around the isolated ascending aorta for indirect assessment of pulmonary blood flow. Ischemia was induced by clamping the left pulmonary artery and the left main bronchus with two microvascular clips (Biemer-Clip, Aesculap, Tuttlingen, Germany). During the ischemic time period, the left lung was kept inflated and the opened chest was covered with wet gauze. The right lung was ventilated with room air and a tidal volume of 2.0 mL at a frequency of 80 minutes⫺1. Positive end-expiratory pressure was kept constant at 2 cm H2O. After ischemia for 50 minutes, the left lung was reperfused and ventilated for 120 minutes or until the death of the animal. After the first 10 minutes of reperfusion, the right pulmonary artery was ligated and the right main bronchus was clamped to exclude the nonischemic right lung from reperfusion.

Experimental Groups The animals were divided into three groups (n ⫽ 7 per group). All animals received a bolus injection of heparin (100 IU/kg) before catheterization of the pulmonary arterial trunk. In addition, animals intraarterially received either heparin (heparin-sodium, Sigma, Deisenhofen, Germany; 200 IU/kg ⫽ 1.1 mg/kg; n ⫽ 7) or the nonanticoagulant NA heparin (Sigma, Deisenhofen, Germany; 1.1 mg/kg; n ⫽ 7) before ischemia. Animals that received saline were the control group (n ⫽ 7). The dosage of 100 IU/kg heparin equals that given during major oper-

Mean systemic arterial blood pressure (MAP, mm Hg), mean pulmonary arterial blood pressure (MPAP, mm Hg), and blood flow through the ascending aorta (BF, mL/min) were recorded before ischemia, at the end of ischemia, and at 10, 12, 15, 20, 25, 30, 60, 90, and 120 minutes of reperfusion. Approximate pulmonary vascular resistance (mm Hg/mL ⫻ minutes) was calculated as mean pulmonary arterial blood pressure/BF on the assumption that left atrial pressure is zero and that BF in the ascending aorta represents pulmonary blood flow.

Arterial Blood Analysis Arterial partial oxygen tension (PaO2) was analyzed with a gas analyzer (Model 348, Chiron Diagnosis, Fernwald, Germany) before ischemia, at the end of ischemia, and at 15, 30, 60, 90, and 120 minutes of reperfusion. Hematocrit (percent) was measured before ischemia and at the end of ischemia with the blood gas analyzer. White blood cells and platelets were counted before ischemia with an automatic cell counter (Act Diff, Coulter Electronic, Krefeld, Germany).

Histologic Examinations At the end of the experiment, phosphate-buffered 4% formalin was instilled intratracheally into the left lung by gravity of 20 cm H2O. The lung was further immersed in formalin, embedded in paraffin, and stained with hematoxylin-eosin or methylene blue azur 2. The degree of alveolar edema, alveolar exudate, and alveolar hemorrhage, as well as the number of polymorphonuclear leukocytes in alveoli, red blood cells (RBCs) in alveolar capillaries, and polymorphonuclear leukocytes in alveolar capillaries were scaled semiquantitatively (grade: 0 ⫽ no, 1 ⫽ mild or little, 2 ⫽ moderate, 3 ⫽ severe or much).

Statistical Analysis All data are expressed as the mean ⫾ standard error of the mean. Survival of the animals at 120 minutes of reperfusion was analyzed by Fisher’s exact probability test. Parametric data were evaluated by one-way analysis of variance and Scheffe’s multiple comparison test. Semiquantitative scoring of lung tissue specimen was analyzed with Kruskal-Wallis one-way analysis of variance on Ranks and Dunn’s multiple comparison test. A p value less than 0.05 was considered significant. Analyses were performed with Stat View (Abacus Concepts, Inc, Berkeley, CA) and SigmaStat (SPSS Inc, Chicago, IL).

Results Body weight, rectal temperature, hematocrit as well as white blood cell and platelet count were within physiologic range and did not differ between the three groups studied. Before ischemia, at the end of ischemia, and at 10

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Fig 1. Blood flow (mL/min) through the ascending aorta at base line, at the end of a 50 minute-period of warm ischemia of the left lung, and during 120 minutes of reperfusion. The nonischemic right lung was excluded from reperfusion after 10 minutes. During the microsurgical procedure all groups received heparin (100 IU/kg). Before ischemia, animals also received either heparin (200 IU/kg; n ⫽ 7), nonanticoagulant N-acetyl (NA) heparin (1.1 mg/kg; n ⫽ 7), or identical volumes of isotonic saline (control, n ⫽ 7). Mean ⫾ standard error: *p ⬍ 0.05 versus control. (B ⫽ baseline, I ⫽ at the end of a 50 minute period of warm ischemia.)

minutes of reperfusion, there were also no significant differences among the groups in BF, PaO2, MAP, and pulmonary vascular resistance (Figs 1– 4).

Animal Survival In the control group, all animals died during the first 70 minutes of reperfusion, thus representing a survival rate

Fig 2. Mean systemic arterial blood pressure (mm Hg) at base line, at the end of a 50 minute-period of warm ischemia of the left lung, and during 120 minutes of reperfusion. The nonischemic right lung was excluded from reperfusion after 10 minutes. During the microsurgical procedure all groups received heparin (100 IU/kg). Before ischemia, animals also received either heparin (200 IU/kg; n ⫽ 7), nonanticoagulant N-acetyl (NA) heparin (1.1 mg/kg; n ⫽ 7), or identical volumes of isotonic saline (control, n ⫽ 7). Mean ⫾ standard error: *p ⬍ 0.05 versus control and **p ⬍ 0.01; #p ⬍ 0.05 versus heparin, and ##p ⬍ 0.01. (B ⫽ baseline, I ⫽ at the end of a 50 minute period of warm ischemia.)

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Fig 3. Pulmonary vascular resistance (mm Hg/mL x min) at base line, at the end of a 50 minute-period of warm ischemia of the left lung, and during 120 minutes of reperfusion. The nonischemic right lung was excluded from reperfusion after 10 minutes. During the microsurgical procedure all groups received heparin (100 IU/kg). Before ischemia the animals also received either heparin (200 IU/kg; n ⫽ 7), nonanticoagulant N-acetyl (NA) heparin (1.1 mg/kg; n ⫽ 7), or identical volumes of isotonic saline (control, n ⫽ 7). Mean ⫾ standard error: *p ⬍ 0.05 versus control. (B ⫽ baseline, I ⫽ at the end of a 50 minute period of warm ischemia.)

of 0 of 7 (Fig 5). In contrast, 6 animals of the heparin group (6 of 7) and all animals treated with NA heparin (7 of 7) survived the total 120 minutes period of reperfusion. Thus survival rate at 120 minutes of reperfusion was significantly higher in the heparin and the NA-heparin group than in the control group (p ⬍ 0.01) (Fig 5). Since most animals of the control group died before 60 minutes of reperfusion, hemodynamic data were statistically analyzed at 30 minutes of reperfusion.

Fig 4. Arterial oxygen partial pressure (mm Hg) at base line, at the end of a 50 minute-period of warm ischemia of the left lung, and during 120 minutes of reperfusion. The nonischemic right lung was excluded from reperfusion after 10 minutes. During the microsurgical procedure all groups received heparin (100 IU/kg). Before ischemia the animals also received either heparin (200 IU/kg; n ⫽ 7), nonanticoagulant N-acetyl (NA) heparin (1.1 mg/kg; n ⫽ 7), or identical volumes of isotonic saline (control, n ⫽ 7). Mean ⫾ standard error: **p ⬍ 0.01 versus control. (B ⫽ baseline, I ⫽ at the end of a 50 minute period of warm ischemia.)

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Histologic Examination

Fig 5. Animal survival during 120 minutes of reperfusion after a 50 minute-period of warm ischemia of the left lung. The nonischemic right lung was excluded from reperfusion after 10 minutes. During the microsurgical procedure all groups received heparin (100 IU/kg). Before ischemia the animals also received either heparin (200 IU/kg; n ⫽ 7), nonanticoagulant N-acetyl (NA) heparin (1.1 mg/kg; n ⫽ 7), or identical volumes of isotonic saline (control, n ⫽ 7). **p ⬍ 0.01 versus control.

Hemodynamic Measurements and Arterial Blood Oxygen Tension The exclusion of the right lung caused an acute decrease of BF in all groups. During subsequent reperfusion, BF recovered in the heparin and the NA-heparin group, but further decreased without recovery in the control group. Significant differences were observed between the control and the two other groups at 30 minutes of reperfusion (p ⬍ 0.05) (Fig 1). Mean systemic arterial blood pressure decreased in all groups upon exclusion of the right lung. However, this decrease was less pronounced in the NA-heparin group (⫺25%) compared with both the control and the heparin group (⫺66% and ⫺58%) (Fig 2). In the NA-heparin group MAP recovered rapidly to base line values within 15 minutes of reperfusion, MAP in the heparin group reached preischemic values after 25 to 30 minutes. In the control group, however, MAP did not recover and remained at low values between 30 to 40 mm Hg (Fig 2). Upon exclusion of the right lung, pulmonary vascular resistance was found to be elevated in all three experimental groups. Pulmonary vascular resistance decreased slowly during the subsequent course of reperfusion in the heparin and the NA-heparin groups, whereas in the control group pulmonary vascular resistance remained increased and was significantly higher than that of the heparin (p ⬍ 0.05) and the NA-heparin group (p ⬍ 0.05) at 30 minutes (Fig 3). The PaO2 remained stable in the range of 95 to 110 mm Hg throughout the 120-minute period of reperfusion in both the heparin and the NA-heparin group, whereas the PaO2 decreased by approximately 50% to values of ⬃50 mm Hg in the control group with significant differences at 15 and 30 minutes of reperfusion when compared to the heparin and the NA-heparin group (p ⬍ 0.01) (Fig 4).

The semiquantitatively graded results of hematoxylineosin stained lung tissue specimen are given in Table 1. The number of RBCs in alveolar capillaries, ie, the quality of nutritive perfusion of capillaries, were significantly higher in the heparin and the NA-heparin groups when compared with the control group. Alveolar edema, exudate, and hemorrhage, as well as the number of polymorphonuclear leukocytes in alveoli and capillaries, all showed minor inflammatory response without significant differences among groups. Methylene blue azur 2 staining of semithin sectioned lung tissue specimen revealed a high number of RBCs in alveolar capillaries, confirming the preservation of nutritive capillary perfusion in both the heparin and the NA-heparin group (Fig 6). In contrast, alveolar walls in the control group were thin and lacking RBCs, indicating capillary no-reflow (Fig 6). Thrombus formation in the pulmonary artery was excluded in all of the animals studied by microdissection of the animals at the end of the experiments.

Comment The pathogenesis of microvascular I/R injury involves two pathophysiologically distinct mechanisms, termed no-reflow and reflow paradox [6 – 8]. Whereas reflow paradox comprises reflow-associated injury, including the release of inflammatory mediators, leukocytic cell activation with interaction and adherence to the microvascular endothelium, and increase of microvascular leakage [8], no-reflow primarily represents ischemia-induced failure of capillary reperfusion [6, 7]. Although several mechanisms have been suggested to Table 1. Semiquantitative Histological Evaluation of Lung Specimens in Animals Subjected to an In Vivo Warm 50Minute Ischemia of the Left Lung Followed by Reperfusion for 120 Minutes Control Alveolar edema Alveolar exudate Alveolar bleeding Polymorphonuclear leukocytes in alveoli Red blood cells in capillaries Polymorphonuclear leukocytes in capillaries

Heparin

N-Acetyl Heparin

0.8 ⫾ 0.2 0.2 ⫾ 0.2 0.8 ⫾ 0.2 0.8 ⫾ 0.2

1.1 ⫾ 0.4 0.4 ⫾ 0.2 1.1 ⫾ 0.1 1.4 ⫾ 0.2

0.4 ⫾ 0.2 0.0 ⫾ 0.0 1.0 ⫾ 0.2 0.7 ⫾ 0.2

1.0 ⫾ 0.0

1.9 ⫾ 0.3a

2.6 ⫾ 0.2a

1.2 ⫾ 0.2

1.7 ⫾ 0.3

1.0 ⫾ 0.2

During microsurgical procedures all groups received heparin (100 IU/kg). Prior to ischemia, animals also received either heparin (200 IU/kg; heparin, n ⫽ 7), nonanticoagulant N-acetyl heparin (1.1 mg/kg; N-acetyl heparin, n ⫽ 7) or identical volumes of isotonic saline (control, n ⫽ 7). Mean ⫾ standard error. No significant differences were found between groups. Kruskal-Wallis analysis of variance on ranks and StudentNewman-Keuls test. Grade: 0 ⫽ no, 1 ⫽ mild or little, 2 ⫽ moderate, 3 ⫽ severe and more. Mean ⫾ standard error. a

p ⬍ 0.05 vs control.

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Fig 6. Semithin sections of lung tissue specimen after a 50 minute-period of warm ischemia followed by 120 minutes of reperfusion. During the microsurgical procedure all groups received heparin (100 IU/kg). Before ischemia the animals also received either heparin (200 IU/kg; n ⫽ 7), nonanticoagulant N-acetyl (NA) heparin (1.1 mg/kg; n ⫽ 7), or identical volumes of isotonic saline (control, n ⫽ 7). The thin alveolar walls lacking red blood cells indicate failure of capillary reperfusion in the nontreated control group (A), whereas, note the preserved capillary perfusion in the heparin group (B) and the NA-heparin group (C). (Methylene blue azur 2 staining, magnification ⫻ 400.)

promote postischemic no-reflow of reperfused organs (eg, intravascular thrombus formation, intravascular hemoconcentration, endothelial cell swelling, interstitial edema formation, hypoxia-induced vasoconstriction, capillary plugging by leukocytes) [7], the determinants of the no-reflow phenomenon, as well as its relevance in the manifestation of postischemic lung injury, are not completely elucidated yet. In the present study, both heparin and NA heparin were protective in the experimental setting of warm I/R injury of the lung with postischemic restoration of hemodynamics and maintenance of adequate oxygenation. After 50 minutes ischemia followed by 120 minutes of reperfusion, lungs did not exhibit an extensive inflammatory response as indicated by only moderate microvascular leukocyte accumulation and minor tissue edema formation. Heparin-treated and NA-heparin-treated animals did not differ in the extent of inflammatory lung injury from that in control animals. Marked differences, however, were found in the RBC-reperfusion of alveolar capillaries. Abundant RBCs were found located in alveolar capillaries of heparin-treated and NA-heparintreated animals, while postischemic lung tissue specimen in nontreated controls failed to show capillary reperfusion. This preservation of nutritive capillary perfusion might, at least in part, explain the protection against postischemic lung injury, as observed in the heparin and the NA-heparin group. Absence of capillary reperfusion in the control animals might be causative for the slightly, but insignificant, lower values of alveolar edema and alveolar exudates (Table 1), because capillary reflow is a prerequisite for the occurrence and manifestation of these inflammatory features of postischemic reperfusion injury. Preserving the normal homeostasis of the endothelium and maintaining the balance between the vasoactive mediators released by the endothelium is critical for the prevention of endothelial dysfunction and nutritive perfusion failure during I/R injury. Apart from coagulation, heparin can modulate a variety of biological processes, such as inhibition of the activation of polymorphonuclear leukocytes [9] and components of the complement system [10]. Moreover, heparin oligosaccharides, including

nonanticoagulant tetrasaccharides, have been shown to directly block L-selectin and P-selectin, thereby inhibiting microvascular leukocyte accumulation during acute inflammation [11]. Heparin further protects the microvascular endothelium against free radical damage [12] and preserves its nitric oxide activity [13, 14]. The Nacetyl heparin differs from the commercial heparin in that it lacks anticoagulant activity, but like heparin it is able to modulate or prevent the activation of complement [5, 10]. The structure of NA heparin has an acetyl group placed instead of sulfate, which inhibits its binding to antithrombin III. So basically the fraction of heparin is the same length as regular heparin with only the substitution of the acetyl group. In accordance with studies demonstrating that heparin maintains microvascular patency in the liver during and after low-flow shock state [15–17], we could observe preserved capillary perfusion in reperfused postischemic lungs after pretreatment with both heparin and nonanticoagulant NA heparin. Thus this protection seems to not be the result of the anticoagulative activity of heparin, but may rather be the result of the direct effect of the negatively charged nature of heparin itself. The high electronegative charge of heparin keeps erythrocytes and, perhaps, all other blood cells dispersed [18], probably also during ischemia-associated standstill of pulmonary perfusion. This effect might even be enhanced by an increase in the negative charge of the endothelium as a result of absorption of heparin [19]. The important role of the negatively charged endothelial cell surface in maintaining the integrity of the alveolocapillary membrane has been appreciated by experiments showing increased pulmonary vascular permeability by heparinase-induced removal of the negatively charged heparan sulfates in the glycocalyx [20]. These negative charges may function as a physiologically significant charge barrier to the transvascular movement of molecules and to the prothrombotic platelet-leukocyteendothelial cell interaction after warm I/R of the lung [21]. The additional capability of heparin to decrease blood viscosity [19] might further improve patency in the postischemic pulmonary microcirculation, as observed in the present study by the obvious reperfusion of alveolar

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capillaries by RBCs. As to whether pulmonary microvascular protection is also caused by the heparin-mediated vasorelaxation through the NO pathway, as has been shown in coronary I/R injury [13, 14], cannot be deduced from the present results. However, the importance of the NO-cGMP pathway in protection against lung injury is clearly underlined by studies demonstrating the attenuation of I/R injury of the lung by application of the NO-donor L-arginine [22]. Although our present study indicates that administration of heparin and nonanticoagulant NA heparin provides protection against I/R injury of the lung, the precise mechanisms by which these heparins act still remain unknown. However, these data may prove clinically significant, allowing the administration of nonanticoagulant heparin derivatives, such as NA heparin to preserve lung function and survival after episodes of pulmonary I/R. In view of the relevant risk of hemorrhagic complications with the unmodified heparin [23], the use of nonanticoagulant heparin may have clinical relevance, because this approach could minimize or prevent pulmonary injury in the absence of the adverse complications of bleeding.

8. 9.

10. 11.

12. 13.

14.

15. Brigitte Vollmar is recipient of a Heisenberg-Stipendium of the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, Germany (V0 450/6 –1).

References 1. Wright JG, Kerr JC, Valeri CR, Hobson RW. Heparin decreases ischemia-reperfusion injury in isolated canine gracilis model. Arch Surg 1988;123:470–2. 2. Saliba MJ, Covell JW, Bloor CM. Effects of heparin in large doses on the extent of myocardial ischemia after acute coronary occlusion in the dog. Am J Cardiol 1976;37:599 – 604. 3. Sternbergh WC III, Makhoul RG, Adelman B. Heparin prevents postischemic endothelial cell dysfunction by a mechanism independent of its anticoagulant activity. J Vasc Surg 1993;17:318–27. 4. Sternbergh WC III, Sobel M, Makhoul RG. Postischemic endothelial cell dysfunction is attenuated by a novel nonanticoagulant heparin. Surg Forum 1992;43:343–5. 5. Fryer A, Huang YC, Rao G, et al. Selective O-desulfation produces nonanticoagulant heparin that retains pharmacological activity in the lung. J Pharmacol Exp Ther 1997;282: 208–19. 6. Menger MD, Steiner D, Messmer K. Microvascular ischemia-reperfusion injury in striated muscle: significance of “no reflow.” Am J Physiol 1992;263:H1892–900. 7. Menger MD, Rucker M, Vollmar B. Capillary dysfunction in

16. 17.

18.

19. 20.

21. 22. 23.

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striated muscle ischemia/reperfusion: on the mechanisms of capillary “no-reflow.” Shock 1997;8:2–7. Menger MD, Pelikan S, Steiner D, Messmer K. Microvascular ischemia-reperfusion injury in striated muscle: significance of “reflow paradox.” Am J Physiol 1992;263:H1901– 6. Laghi-Pasini F, Pasqui AL, Ceccetalli L, Capecchi PL, Orrico A, DiPerri T. Heparin inhibition of polymorphnuclear leukocyte activation in vitro. A possible pharmacological approach to granulocyte-mediated vascular damage. Thromb Res 1984;35:527–37. Weiler JM, Edens RE, Linhardt RJ, Kapelanski DP. Heparin and modified heparin inhibit complement activation in vivo. J Immunol 1992;148:3210–5. Nelson RM, Cecconi O, Roberts WG, Aruffo A, Linhardt RJ, Bevilacqua MP. Heparin oligosaccharides bind L- and Pselectin and inhibit acute inflammation. Blood 1993;82: 3253– 8. Hiebert LM, Liu JM. Heparin protects cultured arterial endothelial cells from damage by toxic oxygen metabolites. Atherosclerosis 1990;83:47–51. Kouretas PC, Myers AK, Kim YD, et al. Heparin and nonanticoagulant heparin preserve regional myocardial contractility after ischemia-reperfusion injury: role of nitric oxide. J Thorac Cardiovasc Surg 1998;115:440– 8. Kouretas PC, Kim YD, Cahill PA, et al. Nonanticoagulant heparin prevents coronary endothelial dysfunction after brief ischemia-reperfusion injury in the dog. Circulation 1999;99:1062– 8. Rana MW, Singh G, Wang P, Ayala A, Zhou M, Chaudry IH. Protective effects of preheparinization on the microvasculature during and after hemorrhagic shock. J Trauma 1992;32: 420– 6. Wang P, Singh G, Rana MW, Ba ZF, Chaudry IH. Preheparinization improves organ function after hemorrhage and resuscitation. Am J Physiol 1990;259:R645–50. Wang P, Ba ZF, Reich SS, Zhou M, Holme KR, Chaudry IH. Effects of nonanticoagulant heparin on cardiovascular and hepatocellular function after hemorrhagic shock. Am J Physiol 1996;270:H1294 –302. Srinivasan S, Aaron R, Chopra PS, Lucas T, Sawyer PN. Effect of thrombotic and antithrombotic drugs on the surface charge characteristics of canine blood vessels: in vivo and in vitro studies. Surgery 1968;64:827–33. Coon WW, Willis PW. Some side effects of heparin, heparinoids, and their antagonists. Clin Pharmacol Therap 1966;7: 379–98. Sunnergren KP, Fairman RP, DeBlois GG, Glauser FL. Effects of protamine, heparinase, and hyaluronidase on endothelial permeability and surface charge. J Appl Physiol 1987; 63:1987–92. Chang SW, Voelkel NF. Charge-related lung microvascular injury. Am Rev Respir Dis 1989;139:534– 45. Yoshida K, Yoshimura K, Haniuda M. L-arginine inhibits ischemia-reperfusion injury in rabbits. J Surg Res 1999;85: 9–16. Kelton JG, Hirsh J. Bleeding associated with antithrombotic therapy. Semin Hematol 1980;17:259–91.

DISCUSSION DR JOSEPH B. ZWISCHENBERGER (Galveston, TX): Dr Vollmar, you have educated us that heparin is a complex mucopolysaccharide macromolecule that exhibits influences on both the cellular elements and the mediators of inflammation, and most intriguing is the fact that your study targeted the capillary no-reflow phenomenon. You performed histology and showed us red cell reperfusion, but you didn’t tell us whether there was a characteristic of the cellular reperfusion

elements that may give us some idea of the mechanism of how N-acetyl heparin may have influenced the inflammatory process. DR VOLLMAR: Actually we looked specifically at the polymorphonuclears by staining with chloroacetate esterase, and we didn’t see any significant differences between the three groups. So there was a very moderate inflammatory response, and we

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are speculating the 100 units of conventional heparin might be beneficial that there were no differences in terms of inflammatory response between the three groups. DR ZWISCHENBERGER: If you attribute this to the negative charge alone, is there a less complex molecule to test than N-acetyl heparin? Are there alternatives or other choices which may have similar properties that don’t have all the complexities of heparin per se? DR VOLLMAR: There might be alternatives. Actually we are that trusting in our hypothesis that it is the negative charge, because there is literature in coronary ischemia and reperfusion that with protamine, which antagonizes or neutralizes heparin, you can ameliorate those effects. So this is

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indirect evidence that it is due to the negative charge. I never thought about alternatives because the N-acetyl heparin does not have any of these maybe negative effects like heparin does. So I do not see any complication by giving that drug. DR ZWISCHENBERGER: We have known in the adult respiratory distress syndrome models that heparin can reverse the development of adult respiratory distress syndrome for years, but because of the anticoagulant properties, it has been avoided in clinical use. DR VOLLMAR: Right. DR ZWISCHENBERGER: This is an exciting new prospect. Thank you.

INVITED COMMENTARY This laboratory study is a significant addition to our evolving understanding of the ischemia/reperfusion response. In our minds it has clinical relevance even as a lab effort. The authors employed delicate microsurgical techniques to effect an elegant rodent model of single lung transplantation. Their mechanism of study seems sound and their results should be similar in other models. Assuming the protective properties of Heparin and N-acetyl-heparin are demonstrated by others in similar models, one could easily translate these results to the clinical arena. One concern is that N-acetyl-heparin indeed exhibits no anticoagulant properties. Dr Nakamura and colleagues’ most basic finding was that of no-reflow in the control group receiving no heparin substrate at all. If the non-anticoagulant heparin did have anticoagulant activity (something they did screen for) these results could have been explained on that basis. Realizing they did not at autopsy find any “gross thrombosis” may not fully satisfy our concern over this potential issue. Future studies should contain data as to the coagulation status at the various stages of the experiment. If this concern is indeed invalid then this paper warrants a real change in our thinking. Our algorithm does not always include heparin since the donor receives 30K or 40K units, which probably prevents graft thrombosis.

© 2001 by The Society of Thoracic Surgeons Published by Elsevier Science Inc

This concern over postoperative bleeding has led us at times to avoid heparin for single lung transplant cases that do not require cardiopulmonary bypass (CPB). Dr Nakamura and associates has shown us that there may well be another benefit of heparin we have overlooked. In fact, most of our single or double single lung transplants that do not require CPB have not had bleeding associated with heparin as much as a lack of prolene, surgical clips, or a diffuse coagulopathy associated with a technically difficult implantation. If these findings are valid, we should use heparin on all of our patients and simply do a better job of drying them up. Further study to help us with necessary dosing and length of dosing for maximum avoidance of the ischemia/reperfusion response is needed as well as clinical corroboration. John H. Calhoon, MD Scott B. Johnson, MD Edward Y. Sako, MD, PhD Department of Thoracic Surgery University of Texas Health Science Center 7703 Floyd Curl Dr San Antonio, TX 78284-3900 e-mail: [email protected]

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