| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Ann Thorac Surg 2001;72:1183-1189
© 2001 The Society of Thoracic Surgeons
a Department of Thoracic and Cardiovascular Surgery, Homburg/Saar, Germany
b Institute for Clinical and Experimental Surgery, University of Saarland, Homburg/Saar, Germany
c Department of Thoracic Surgery, Kyoto University, Kyoto, Japan
Address reprint requests to Dr Vollmar, Institute for Clinical and Experimental Surgery, University of Saarland, 66421 Homburg/Saar, Germany
e-mail: exbvol{at}med-rz.uni-sb.de
Presented at the Thirty-seventh Annual Meeting of The Society of Thoracic Surgeons, New Orleans, LA, Jan 29–31, 2001.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
|---|
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 = 7), NA heparin (1.1 mg/kg; n = 7), or saline (control, n = 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 NA-heparin 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.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
|---|
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.
|
Material and methods |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
|---|
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 24G-catheter (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 operations, whereas the dosage of 300 IU/kg heparin is used in cardiopulmonary bypass procedures.
Hemodynamic measurements
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 x 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 |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
|---|
|
|
|
|
|
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).
Histologic examination
The semiquantitatively graded results of hematoxylin-eosin 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 |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
|---|
Although several mechanisms have been suggested to 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-heparin-treated 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 N-acetyl 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-leukocyte-endothelial 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 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.
|
Acknowledgments |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
|---|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
|---|
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 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 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.
|
References |
|---|
|
TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsDiscussionReferences |
|---|
Related Article
This article has been cited by other articles:
|
|
 |
|
  G. Szabo, L. Seres, P. Soos, M. Gorenflo, B. Merkely, F. Horkay, M. Karck, and T. Radovits Tetrahydrobiopterin improves cardiac and pulmonary function after cardiopulmonary bypass Eur J Cardiothorac Surg, September 1, 2011; 40(3): 695 - 700. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ANN THORAC SURG | ASIAN CARDIOVASC THORAC ANN | EUR J CARDIOTHORAC SURG |
| J THORAC CARDIOVASC SURG | ICVTS | ALL CTSNet JOURNALS |
