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Ann Thorac Surg 1998;65:935-938
© 1998 The Society of Thoracic Surgeons

Nitric Oxide Potentiates Acute Lung Injury in an Isolated Rabbit Lung Model

Constanze S. Rayhrer, MDa, Timothy D. Edmisten, MDa, Gerald A. Cephas, MDa, Curtis G. Tribble, MDa, Irving L. Kron, MDa, Jeffrey S. Young, MDa

a Department of Surgery, University of Virginia Health Sciences Center, Charlottesville, Virginia, USA

Accepted for publication December 3, 1997.

Address reprint requests to Dr Young, Department of Surgery, University of Virginia Health Sciences Center, Box 10005, Charlottesville, VA 22906-0005
e-mail: (jsy2b{at}virginia.edu)


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 References
 
Background. The effect of inhaled nitric oxide (NO) treatment on pulmonary function in the setting of adult respiratory distress syndrome is controversial. We examined the effect of inhaled NO on pulmonary function in an isolated rabbit lung model of oleic acid (OA)-induced acute lung injury. We hypothesized that NO would decrease pulmonary artery pressure and improve oxygenation.

Methods. Rabbit heart-lung blocks were isolated, flushed in vivo, harvested, and immediately perfused with whole blood and ventilated with 50% oxygen (O2). Pulmonary artery pressure was determined every 15 seconds for 60 minutes of perfusion. Oxygenation was determined by blood gas analysis of pulmonary venous effluent at 0, 20, 40, and 60 minutes after initiation of OA infusion. Rabbits were randomized into four study groups: saline control; OA control, which received a 20-minute infusion of 50% OA/ethanol solution; NO treatment (20 ppm NO inhaled before OA infusion); and NO control, which underwent NO (20 ppm) pretreatment, followed by saline infusion. Pulmonary artery pressure, oxygenation (arteriovenous O2 difference), compliance, and wet/dry lung weight were determined.

Results. Pretreatment with NO caused significant increases in pulmonary artery pressure (NO treatment versus NO control and saline control; no significant difference between NO treatment group and OA control group), and did not improve oxygenation in our model.

Conclusions. Contrary to our hypothesis, pretreatment with NO potentiates acute lung injury in our isolated lung model. There was significant exacerbation of pulmonary hypertension and no improvement in oxygenation. Further investigation of the possible deleterious effects of NO in acute lung injury are needed, especially in the early acute phases of this process.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 References
 
The effect of inhaled nitric oxide (NO) treatment on pulmonary function in the setting of adult respiratory distress syndrome (ARDS) is controversial. Although studies have shown an increase in oxygenation and a decrease in pulmonary vascular resistance, it has been difficult to demonstrate an improvement in mortality in clinical studies [1–6]. In addition, the effects of prophylactic treatment in patients at high risk for the development of ARDS are unknown. The difficulties in isolating the clinical actions of NO in ARDS may be related to its many physiologic actions. Nitric oxide is thought to decrease hypoxia and pulmonary hypertension by several mechanisms. One known mechanism is pulmonary vasodilatation, which in turn decreases pulmonary shunting and improves oxygenation [1]. Nitric oxide also may improve oxygenation by reducing pulmonary hypertension and reducing the hydrostatic pressure within the pulmonary vascular bed, thus decreasing the formation of pulmonary edema [2]. It has been suggested that NO reduces neutrophil activation and expression of adhesion molecules, which may decrease pulmonary inflammation [7].

Adult respiratory distress syndrome is associated with pulmonary hypertension, intrapulmonary shunting, increased microvascular permeability leading to pulmonary edema, and compromised oxygen diffusion. Intravenous administration of oleic acid (OA), a polyunsaturated fatty acid, produces a reproducible acute lung injury (ALI) mimicking ARDS. As in ARDS, OA injury is associated with pulmonary hypertension, microvascular cellular aggregation and obstruction, intrapulmonary shunting, and microvascular permeability. We hypothesized that pretreatment with inhaled NO would result in improved oxygenation and pulmonary function in our model.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 References
 
Our experimental protocol was approved by the University of Virginia’s Animal Research Committee. All animals were cared for in accordance with Committee guidelines, as well as those set forth by the National Institutes of Health.

New Zealand White rabbits were anesthetized with intramuscular injections of xylazine (5 mg/kg) and ketamine (50 mg/kg). After anesthesia, blood was obtained from systemically heparinized (500 units/kg intravenously) donor rabbits via right ventricular cannulation. This blood was diluted with normal saline solution to a hematocrit of 25% to 30%. Study rabbits weighing between 3 and 3.5 kg received tracheostomies after administration of vecuronium. Rabbits were ventilated with a small-animal ventilator (Kent Scientific) to a respiratory rate of 20 breaths/min and tidal volume of 10 mL/kg. A fixed inspired concentration of oxygen of 50% was maintained throughout the experiment.

A midline thoracotomy was performed after tracheostomy, and the animals were systemically heparinized. After isolation and ligation of the two superior venae cavae and single inferior vena cava, the pulmonary artery was cannulated via the right heart. A 6°C normal saline solution flush was then administered through the pulmonary artery catheter (total volume, 40 mL/kg). Outflow control was obtained by placing dual catheters through the left ventricle to the left atrium. Thymectomy was then performed. The lung/heart block was removed from the chest and placed ex vivo into our continuous blood perfusion circuit. Lungs were allowed to stabilize after the initiation of perfusion for 2 to 4 minutes before measurements were begun. Lungs with evidence of air leak were excluded from the study. Blood was circulated through the circuit at 60 mL/min with a Masterflex roller pump (Cole-Parmer Instruments).

Nitric oxide was delivered into the ventilatory circuit directly after passing through an NO sensor. Mixing of NO and O2 occurred just before entry into the ventilator’s intake to minimize radical formation. Nitric oxide at 20 ppm was delivered to two groups from the time of tracheostomy and continued throughout the 60-minute study period.

Oleic acid injury was induced with 0.4 mL of a solution of 50% OA and 95% ethanol delivered by a micropump over 20 minutes directly into the perfusion circuit. Control groups received an equal amount of normal saline solution delivered in a similar manner (Fig 1).



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  		Fig 1. Experimental apparatus used. (ABG = arterial blood gas measurement site; NO = nitric oxide; OA = oleic acid; SNP = sodium nitroprusside; vent = ventilator.)

 
Measurement of pulmonary artery pressure and pulmonary resistance was made at 15-second intervals. Lung edema was calculated by examining sections of lower lobes, and was recorded as wet/dry weight ratios. The oxygenation capacity of each lung was calculated at 20-minute intervals for 1 hour by measuring arterial blood gases of delivered fresh venous whole blood obtained from a reservoir before and after perfusion through the lung (see Fig 1). Sections of lower lobes were preserved in formalin for histologic examination.

Study design
Thirty-two rabbits were divided into four equal groups. The saline control (SC) group received normal saline injection as described. An NO control (NOC) group received saline injection and administration of NO at 20 ppm. An OA control group (OAC) received 20 minutes of continuous OA administration without NO. The NO treatment (NORx) group received 20 minutes of OA injury and continuous treatment with NO.

Analysis
Data analysis of pulmonary artery pressure, compliance, pulmonary vascular resistance, and arteriovenous oxygenation difference was performed by comparing the percent change from initial values to those at 20, 40, and 60 minutes. Wet/dry weight ratios were calculated from portions of lower lobes from each animal. Resulting values were compared using analysis of variance and reported as the mean ± standard error of the mean. A p value less than 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 References
 
A total of 31 lung-heart blocks were analyzed. The percent change of each parameter during the study period was compared between control and NO treatment groups. At 60 minutes, the changes in pulmonary artery pressure in the SC (-1.6% ± 3.2%) and NOC (-5.2% ± 6.6%) groups were significantly less (p < 0.05) than in the NORx (36.4% ± 13.9%) and the OAC (24.2% ± 10.7%) groups (Fig 2). It should be noted that even though significance was not reached, pulmonary artery pressure was increased to a greater extent in the NORx group than in the OAC group. No significant differences in compliance were noted between groups until 60 minutes (Fig 3). Changes in NOC and OAC compliance (1.31% ± 3.12%, -6.59% ± 8.14%) were both significantly less than in the NORx study group (-19.67% ± 5.21%, p < 0.01). There were no significant differences in pulmonary vascular resistance (Fig 4) or oxygenation capacity (Fig 5) among any of the groups. There was a significant difference in wet/dry weight ratios between the SC and NOC groups and the OAC group (Fig 6). The OAC and NORx groups had consistently higher pulmonary vascular resistance and wet/dry ratios than the SC or NOC groups (Table 1).



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  		Fig 2. Pulmonary artery pressure (PAP) measurements (mm Hg) at 20, 40, and 60 minutes after initiation of oleic acid (OA) infusion or saline infusion. (NOC = nitric oxide control; NORx = nitric oxide treatment.)

 


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  		Fig 3. Changes in dynamic airway compliance (CPL) at 20, 40, and 60 minutes after initiation of saline or oleic acid (OA) infusion. (NOC = nitric oxide control; NORx = nitric oxide treatment.)

 


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  		Fig 4. Changes in pulmonary vascular resistance (PVR; dyne · s/cm5) at 20, 40, and 60 minutes after initiation of oleic acid (OA) or saline infusion. (NOC = nitric oxide control; NORx = nitric oxide treatment.)

 


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  		Fig 5. Changes in arteriovenous O2 difference (AVO2; mm Hg) at 20, 40, and 60 minutes after initiation of saline or oleic acid (OA) infusion. (NOC = nitric oxide control; NORx = nitric oxide treatment.)

 


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  		Fig 6. Wet-to-dry weight ratios for each experimental group. (NOC = nitric oxide control; NORx = nitric oxide treatment; OAC = oleic acid control; SC = saline control; *p < 0.05 versus OAC.)

 

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  		Table 1. Experimental Data Expressed as Percent Change From Baseline to 60 Minutes

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
References
 
Analysis of the therapeutic role of NO in ARDS has been an area of active investigation. A variety of studies have shown that NO decreases pulmonary vascular resistance and pulmonary wedge pressures [8–11]. Other actions of NO include inhibition of platelet aggregation [12] and reduction in hydrogen peroxide formation and beta-2 integrin expression, as well as interleukin-6 and -8 production [7]. Despite these apparent positive effects, consistent survival advantages in patients treated with NO have been difficult to demonstrate [9–11].

Consistent improvement in physiologic and oxygenation parameters also have been difficult to demonstrate in various animal models. Eichenger and Walker [13] found that NO treatment did not affect lung fluid flux in rat lungs. Eppinger and associates [14] found that NO was toxic early in reperfusion, but was protective at 4 hours, probably because of reversal of postischemic lung hypoperfusion and reduction of lung neutrophil sequestration. In models of OA-induced lung injury, Putenson and colleagues [15] found that whereas NO inhalation lowered pulmonary vascular resistance, recruitment of gas exchange units with continuous positive airway pressure was needed to improve oxygenation. Shah and coworkers [16] demonstrated that NO caused a dose-dependent reduction in pulmonary pressures but had no effect on extravascular lung water.

Our experimental model has been used to examine the effects of surfactant, interleukin-2 antagonism, and thromboxane receptor antagonists on OA-induced lung injury and ischemia/reperfusion injury in isolated rabbit lungs [17]. Our model allows us to examine the pulmonary effects of a process without interference from other organ systems.

In this series of experiments, we found that NO treatment, concurrent with OA injury, worsened pulmonary hypertension, did not improve oxygenation, and significantly decreased compliance when compared with saline and NO controls. This is similar to the results found in Eppinger and associates’ [14] work and represents another aspect of NO actions that may be clinically important. Nitric oxide’s vasodilatory effects in the early stages of ALI and reperfusion may allow infusion of harmful mediators into pulmonary vasculature previously protected by vasoconstriction and redirection of pulmonary blood flow. In this instance, pretreatment with NO possibly allowed OA into segments of the lung that would have been protected. This would cause increased pulmonary damage by OA that overshadowed the beneficial effects of NO. In fact, this interpretation accurately reflects our findings. Clinically, treatment with inhaled NO early in the course of septic or reperfusion ALI may allow more of the pulmonary vasculature to come into contact with harmful mediators, increasing the extent of ALI and worsening pulmonary function. The beneficial effect of NO in ARDS is likely caused by vasodilatation of aerated segments, thus redirecting blood flow to functioning pulmonary units, decreasing shunt fraction, and improving oxygenation. This effect, in most studies, occurs after ARDS is clinically evident, and thus it is unlikely that ongoing injury is present. We believe our data should caution against early or preemptive treatment of ALI with inhaled NO.

In conclusion, OA produced an ALI in this isolated rabbit lung/heart model similar to ARDS with increased pulmonary artery pressure, decreased compliance and oxygenation, and pulmonary edema. Although NO did not change oxygenation, it did potentiate changes in pulmonary artery pressure and compliance. Both parameters indicated worsened ALI after 60 minutes of study. We conclude that, in this model, pretreatment with NO potentiated ALI in the acute phase. Although deteriorating lung function was evident, the mechanism was not clear. We hypothesize that the vasodilatory effects of NO during OA administration may have had the untoward effect of increasing delivery of OA to uninjured alveoli.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 References
 

  1. Zwissler B., Kemming G., Habler O., et al. Inhaled prostacyclin (PGI2) versus inhaled nitric oxide in adult respiratory distress syndrome. Am J Respir Crit Care Med 1996;154:1671-1677.[Abstract]
  2. Rossetti M., Guenard H., Gabinski C. Effects of nitric oxide inhalation on pulmonary serial vascular resistances in ARDS. Am J Respir Crit Care Med 1996;154:1375-1381.[Abstract]
  3. Andresen M., Dougnac A., Castillo L., et al. Prolonged inhalation of nitric oxide in adult respiratory distress syndrome in a case. Rev Med Chile 1996;124:94-102.[Medline]
  4. Demirakca S., Dotsch J., Knothe C., et al. Inhaled nitric oxide in neonatal and pediatric acute respiratory distress syndrome: dose response, prolonged inhalation, and weaning. Crit Care Med 1996;24:1913-1919.[Medline]
  5. Betbese A.J., Perez M., Bak E., Ballus J., Net A., Mancebo J. Gas exchange and hemodynamic effects of nitric oxide inhalation in patients with acute respiratory distress syndrome. Med Clin 1996;107:201-206.
  6. Krafft P., Fridrich P., Fitzgerald R.D., Koc D., Steltzer H. Effectiveness of nitric oxide inhalation in septic ARDS. Chest 1996;109:486-493.[Abstract/Free Full Text]
  7. Chollet-Martin S., Gatecel C., Kermarrec N., Gougerot-Pocidalo M.A., Payen D.M. Alveolar neutrophil functions and cytokine levels in patients with the adult respiratory distress syndrome during nitric oxide inhalation. Am J Respir Crit Care Med 1996;153:985-990.[Abstract]
  8. Lowson S.M., Rich G.F., McArdle P.A., Jaidev J., Morris G.N. The response to varying concentrations of inhaled nitric oxide in patients with acute respiratory distress syndrome. Anesth Analg 1996;82:574-581.[Abstract]
  9. McIntyre R.C., Jr, Moore F.A., Moore E.E., et al. Inhaled nitric oxide variably improves oxygenation and pulmonary hypertension in patients with acute respiratory distress syndrome. Trauma 1995;39:418-425.[Medline]
  10. Rossaint R., Slama K., Steudel W., et al. Effects of inhaled nitric oxide on right ventricular function in severe acute respiratory distress syndrome. Intensive Care Med 1995;21:197-203.[Medline]
  11. Rossaint R., Gerlach H., Schmidt-Ruhnke H., et al. Efficacy of inhaled nitric oxide in patients with severe ARDS. Chest 1995;107:1107-1115.[Abstract/Free Full Text]
  12. Samama C.M., Diaby M., Fellahi J.L., et al. Inhibition of platelet aggregation by inhaled nitric oxide in patients with acute respiratory distress syndrome. Anesthesiology 1995;83:56-65.[Medline]
  13. Eichinger M.R., Walker B.R. Nitric oxide and cGMP do not affect fluid flux in isolated rat lungs. J Appl Physiol 1996;80:69-76.[Abstract/Free Full Text]
  14. Eppinger M.J., Ward P.A., Jones M.L., Bolling S.F., Deeb G.M. Disparate effects of nitric oxide on lung ischemia-reperfusion injury. Ann Thorac Surg 1995;60:1169-1176.[Abstract/Free Full Text]
  15. Putensen C., Rasanen J., Lopez F.A., Downs J.B. Continuous positive airway pressure modulates effect of inhaled nitric oxide on the ventilation-perfusion distributions in canine lung injury. Chest 1994;106:1563-1569.[Abstract/Free Full Text]
  16. Shah N.S., Nakayama D.K., Jacob T.D., et al. Efficacy of inhaled nitric oxide in a porcine model of adult respiratory distress syndrome. Arch Surg 1994;129:158-164.[Abstract/Free Full Text]
  17. Theiss S.D., Corbin R.S., Goff C.D., et al. Thromboxane receptor blockade improves oxygenation in an experimental model of acute lung injury. Ann Thorac Surg 1996;61:1453-1457.[Abstract/Free Full Text]



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