Isolated Rat Lung Perfusion Model Research Articles

(868) - Involvement of Rho-Kinase in Lung Ischemia-Reperfusion Injury Pathogenesis: Molecular Analysis in an Isolated Rat Lung Perfusion Model

(868) - Involvement of Rho-Kinase in Lung Ischemia-Reperfusion Injury Pathogenesis: Molecular Analysis in an Isolated Rat Lung Perfusion Model

(737) - Protective Effect of Nebulized Rho-Kinase Inhibitor on Ischemia Reperfusion Injury in Isolated Rat Lung Perfusion Model

(737) - Protective Effect of Nebulized Rho-Kinase Inhibitor on Ischemia Reperfusion Injury in Isolated Rat Lung Perfusion Model

Therapeutic effect of surfactant inhalation during warm ischemia in an isolated rat lung perfusion model

Warm ischemia-reperfusion injury related to donation after cardiac death donors is a crucial and inevitable issue. As surfactant function is known to deteriorate during warm ischemia, we hypothesized that surfactant inhalation during warm ischemia would mitigate warm ischemia-reperfusion injury. We used an isolated rat lung perfusion model. The rats were divided into three groups: sham, control, and surfactant. In the control and surfactant groups, cardiac arrest was induced by ventricular fibrillation. Ventilation was restarted 110 min later; subsequently, the lungs were flushed, and heart and lung block was recovered. In the surfactant group, a natural bovine surfactant Surfacten(®) was inhaled for 3 min at the end of warm ischemia. Then, the lungs were reperfused for 80 min. Surfactant inhalation significantly improved graft functions, effectively increased lung tissue ATP levels, and significantly decreased mRNA levels of IL-6 and IL-6/IL-10 ratio at the end of reperfusion. Histologically, lungs in the surfactant group showed fewer signs of interstitial edema and hemorrhage, and significantly less neutrophilic infiltration than those in the control group. Our results indicated that surfactant inhalation in the last phase of warm ischemia maintained lung tissue energy levels and prevented cytokine production, resulting in the alleviation of warm ischemia-reperfusion injury.

Post-ischemic Infusion of Atrial Natriuretic Peptide Attenuates Warm Ischemia–Reperfusion Injury in Rat Lung

The serious shortage of organs for transplantation, especially lungs, has drawn increasing attention to donation after cardiac death and protection of organs against warm ischemic injury. Atrial natriuretic peptide (ANP) activates guanylate cyclase receptors and increases cyclic guanosine monophosphate (cGMP) levels, which decrease in the lung during ischemia. In this study we investigated the effect on lung ischemia-reperfusion injury of administering synthetic ANP (carperitide) at the onset of reperfusion after warm ischemia. An isolated rat lung perfusion model was used. The rats were allocated into three groups: the control group; the ANP group; and the sham group. In the control and ANP groups, the heart-lung block was exposed to 60 minutes of ischemia at 37 degrees C, and subsequently reperfused for 60 minutes. At the onset of reperfusion, either saline or ANP was added to the perfusate. In the sham group, lungs were continuously perfused without ischemia and only saline was added to the perfusate. ANP significantly reduced pulmonary vascular resistance and pulmonary edema, and improved oxygenation. It also significantly increased cGMP levels in reperfused lungs. Histologically, lungs in the ANP group showed significantly fewer signs of injury and fewer cells demonstrated apoptotic changes or single-stranded DNA than lungs in the control group. Our results indicate that ANP administered at the onset of reperfusion increases cGMP in lung tissue and attenuates warm ischemia-reperfusion injury in isolated perfused rat lung.

Nebulized Phosphodiesterase III Inhibitor During Warm Ischemia Attenuates Pulmonary Ischemia–Reperfusion Injury

The control of warm ischemia-reperfusion injury is crucial in managing donors after cardiac death for lung transplantation. We focused on transalveolar administration as a drug-delivery route for such donors. Milrinone is a phosphodiesterase 3 inhibitor that inhibits the breakdown of cyclic adenosine monophosphate and selectively relaxes smooth muscle. We hypothesized that nebulized milrinone would mitigate warm ischemia-reperfusion injury of lung. This study was conducted with an isolated rat lung perfusion model. Lungs were excised, exposed to 55-minute ischemia at 37 degrees C, and reperfused for 60 minutes. During ischemia, nebulized milrinone (n = 6) or saline (n = 6) was inhaled. Lungs were continuously perfused without ischemia as a sham group (n = 6). Airway resistance, pulmonary vascular resistance, pulmonary compliance, weight gain and blood gas were measured. Adenine nucleotide levels and apoptosis were investigated in the reperfused lungs. Milrinone nebulization decreased post-ischemic pulmonary vascular resistance (0.98 +/- 0.05 and 1.74 +/- 0.17 cm H(2)O/ml.min at 60 minutes of reperfusion in the milrinone and control groups, respectively [p < 0.01]). It did not alter cyclic adenosine monophosphate levels, but it did elevate adenosine triphosphate levels (9.87 +/- 0.38 and 6.91 +/- 0.45 in the milrinone and control groups, respectively [p < 0.01]) and suppressed apoptosis (3.83 +/- 0.91 and 46.17 +/- 3.39 of mean apoptotic cell numbers in the milrinone and control groups, respectively [p < 0.01]). Milrinone nebulization decreased post-ischemic pulmonary vascular resistance, elevated adenosine triphosphate levels, and suppressed apoptosis. Nebulized milrinone has some protective effects against warm ischemia.

446: Successful Subzero Non-Freezing Preservation of Rat Lungs with Supercooling Technology

Purpose: Supercooling is defined as a non-freezing state of liquid below freezing temperature. Stable supercooling at the temperature between 0°C to –10°C is achieved in a newly developed refrigerator with a power voltage of 3,000 V. We hypothesized that supercooling would reduce metabolism of organ grafts and lungs preserved in supercooling would have better physiological function than in the clinically standard storage at 4°C. Methods and Materials: We investigated in a previously published isolated rat lung perfusion model, in which physiological parameters such as tidal volume, arterial oxygen tension, and lung weight were measured. The heart-lung block was preserved for 17 hr and subsequently reperfused for 60 minutes. All animals were divided into three groups. In the supercoolng group (n 7), non-freezing preservation at -2°C was kept in a supercooling refrigerator. In the control group (n 7), the ordinary hypothermic preservation was maintained at 4°C. In the sham group (n 7), after harvesting lungs were continuously perfused without hypothermic preservation. Results: Two animals in the control group were excluded from the following analysis because of severe lung edema within 12 minutes of reperfusion. Oxygen tensions at 60 minutes of reperfusion were 101.8 2.6 in the supercooling, 58.6 10.8 in the control, and 104.4 1.9 in the fresh (mmHg). At 60 minutes, tidal volumes were 2.0 0.1 in the supercooing, 1.0 0.3 in the control, and 2.2 0.1 in the fresh (mL). The lung weight gains during the 60 minutes were 1248 56, 2983 543, and 693 69, in supercooling, in control and in fresh, respectively (mg). Throughout the experiment, oxygen tensions and tidal volumes in the supercooling and the fresh were significantly higher than the control group(p 0.001, p 0.05, respectively), and the lung weight gains in the supercooling and the fresh were significantly lower than the control (p 0.001). Conclusions: Supercooling demonstrated the superiority in hypothermic preservation of rat lungs to the standard storage of 4°C in an isolated rat lung perfusion model.

Protective Effect of a Nebulized β 2-Adrenoreceptor Agonist in Warm Ischemic–Reperfused Rat Lungs

It seems inevitable that non-beating-heart donors will be utilized to resolve the shortage of donors for clinical lung transplantation. The control of warm ischemia-reperfusion injury is crucial in manipulating non-beating-heart donors. We hypothesized that nebulization of a beta2-adrenoreceptor agonist, salmeterol xinafoate (SLM), during warm ischemia would increase lung tissue cyclic adenosine monophosphate (cAMP) levels, resulting in lung protection. Two studies were conducted. The first investigated the effect of SLM nebulization during ischemia on pulmonary ischemia-reperfusion injury, using an isolated rat lung-perfusion model. The heart-lung block was excised with cannulation of the pulmonary artery and vein, exposed to 55 minutes of ischemia at 37 degrees C, and subsequently reperfused for 60 minutes. Several parameters were measured during reperfusion. In the second study, to measure changes in lung tissue cAMP levels during warm ischemia with or without SLM nebulization, rat lungs were harvested and exposed to 60 minutes of warm ischemia with ventilation. Salmeterol xinafoate nebulization significantly decreased the pulmonary shunt fraction, airway resistance, and pulmonary vascular resistance. It also inhibited pulmonary edema throughout the reperfusion period. Lung tissue cAMP was effectively maintained by SLM nebulization at the end of reperfusion. Myeloperoxidase activity in the lungs was decreased significantly by SLM nebulization. Lung tissue cAMP levels decreased during the 60 minutes of warm ischemia, but increased with SLM nebulization (p < 0.01). Our results confirmed that SLM nebulization during warm ischemia maintained lung tissue cAMP levels, resulting in the alleviation of pulmonary warm ischemia-reperfusion injury.

Addition of ATP and MgCl2 to the Preservation Solution Attenuates Lung Reperfusion Injury following Cold Ischemia

Background: In lung transplantation, reperfusion injury following cold ischemia is one of the crucial problems for recipients. Objective: We evaluated the protective effect of adding a combination of ATP and MgCl₂ to the preservation solution against lung reperfusion injury following cold ischemia. Methods: Using an isolated rat lung perfusion model with fresh rat blood as the perfusate, the rats were divided into five groups (n = 6). In the fresh group, the study lungs were flushed with phosphate-buffered saline (PBS), then immediately reperfused for 120 min. In the control group, the study lungs were flushed with PBS, then cold ischemia was induced for 9 h (4°C), after which reperfusion was performed. In the other three groups, the protocols were the same as for the control group except that ATP and/or MgCl₂ were added to the PBS: ATP group (100 µM ATP), MgCl₂ group (100 µM MgCl₂) and ATP + MgCl₂ group (100 µM ATP + 100 µM MgCl₂). Results: In the ATP + MgCl₂ group, the intrapulmonary shunt fraction, peak airway pressure and wet to dry lung weight ratio were significantly lower than those in the control group. No improvement was observed in the ATP or MgCl₂ groups. Histological examination supported these physiological results. In all groups, flush time and lipid peroxide levels in the lungs after cold ischemia did not show any significant differences. Conclusion: The addition of ATP and MgCl₂ to the preservation solution attenuated reperfusion injury following cold ischemia in rat lungs.

"Chemical preconditioning" by 3-nitropropionate reduces ischemia-reperfusion injury in cardiac-arrested rat lungs.

Chemical preconditioning was defined as the induction of resistance to massive disruption of energy metabolism through prior chemical suppression of oxidative phosphorylation, by which phenomena similar to those resulting from increased ischemic tolerance as a result of ischemic preconditioning can be induced. It could be induced by the inhibitor of either mitochondrial complex I or II. We investigated whether or not chemical preconditioning by 3-nitropropionate (an inhibitor of the mitochondrial complex II) can suppress ischemia-reperfusion injury in cardiac-arrested lungs, which will be the major problem in lung transplants donated from non-heart-beating cadavers. In an isolated rat lung perfusion model with fresh rat blood as perfusate, administration of 3-nitropropionate (20 mg/kg) immediately before the induction of cardiac arrest attenuated pulmonary dysfunction during reperfusion after 1 hr postmortem warm ischemia and 1 hr cold preservation. 3-Nitropropionate administration reduced the mitochondrial respiratory functions (state 3 and state 4 respiration, and the respiratory control ratio) before cardiac arrest and kept them at a lower level of activity than when decreased by ischemia alone. 3-Nitropropionate administration also reduced the ATP levels immediately after drug administration. However, 3-nitropropionate did not significantly reduce lipid peroxidation in the lung tissue and mitochondria. These results demonstrated that chemical preconditioning by 3-nitropropionate administration immediately before cardiac arrest suppressed succinate-related oxidation during postmortem warm ischemia and reduced ischemia-reperfusion injury in cardiac arrested rat lungs.

Reperfusion lung injury after cold preservation correlates with decreased levels of intrapulmonary high-energy phosphates.

To evaluate the role of energy state in primary graft dysfunction, which is crucial in lung transplantation, we investigated the relationship between intrapulmonary high-energy phosphate compounds and reperfusion lung injury after cold preservation. Using an isolated rat lung perfusion model with fresh rat blood as perfusate, rat lungs were exposed to various cold preservation periods (0, 6, 9, and 12 hr) and reperfused. We found that extending the preservation period exacerbated the pulmonary hemodynamics after reperfusion. The levels of intrapulmonary high-energy phosphate compounds did not change during cold preservation, but these levels after reperfusion decreased as the preservation period was prolonged. The pulmonary hemodynamics after reperfusion were inversely correlated with the intrapulmonary high-energy phosphate compound levels after reperfusion. Total adenine nucleotide and ATP were sensitive indicators of reperfusion lung injury after cold preservation. Energy charge was not a sensitive indicator. The decreased levels of intrapulmonary high-energy phosphate compounds after reperfusion following cold preservation period were partially caused by their decreased production. These results demonstrated that reperfusion lung injury after cold preservation was closely correlated with decreased levels of intrapulmonary high-energy phosphate compounds after reperfusion, although the levels of the intrapulmonary high-energy phosphate compounds did not change during cold preservation of up to 12 hr.

Comparison of Euro-Collins Solution, Low-Potassium Dextran Solution Containing Glucose, and ET-Kyoto Solution for Lung Preservation in an Extracorporeal Rat Lung Perfusion Model

We have recently developed a modified ET-Kyoto solution by adding N-acetylcysteine, nitroglycerin, and dibutyryl cyclic adenosine monophosphate to the previously reported ET-Kyoto solution. The purpose of this study was to compare the new ET-Kyoto solution with the regular Euro-Collins solution and a low-potassium dextran solution containing glucose (Perfadex^®) in an isolated rat lung perfusion model. Rat lung blocks were preserved with ET-Kyoto solution, with Euro-Collins solution, or with Perfadex for 4 h. Arterial oxygen tension, shunt fraction, peak inspiratory pressure, mean pulmonary arterial pressure, and pulmonary vascular resistance were assessed up to 50 min after reperfusion. ET-Kyoto solution provided a significantly better lung function after reperfusion than Euro-Collins solution and Perfadex, while Perfadex was also superior to the Euro-Collins solution. We conclude that lung preservation with ET-Kyoto solution is significantly superior to Euro-Collins solution and to Perfadex in an isolated rat lung perfusion model.

Involvement of Fibrinogen in Protamine-Induced Pulmonary Hypertension

Protamine reversal of heparin anticoagulation occasionally results in pulmonary hypertension as well as systemic hypotension. To examine the contribution of blood components to this induction of pulmonary hypertension, we developed an isolated rat lung perfusion model and perfused heparinized plasma, heparinized serum, and Hepes (4% bovine serum albumin, 20 mMN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 5 mM glucose, in warm physiological saline) buffer solution with or without fibrinogen. Perfusion with heparinized plasma and Hepes buffer solution with fibrinogen caused pulmonary hypertension; perfusion with heparinized serum or Hepes buffer solution without fibrinogen did not, suggesting that fibrinogen is involved in the induction of pulmonary hypertension. We also labeled protamine with125I and compared the amounts of protamine accumulating in the lung with different concentrations of fibrinogen. The amount of protamine trapped in the lung increased according to the concentration of fibrinogen. Fibrinogen may accelerate the reaction between pulmonary endothelial cells and protamine or protamine–heparin complexes. In the mechanism of protamine-induced pulmonary hypertension, fibrinogen, as well as heparin and protamine, may be an essential component.

Hydroxylamine is a vasorelaxant and a possible intermediate in the oxidative conversion of L-arginine to nitric oxide

Our objective was to determine whether hydroxylamine is a possible intermediate in the oxidative conversion of L-arginine to nitric oxide. Vasorelaxation by hydroxylamine is known to be mediated by nitric oxide. The vasorelaxant properties of hydroxylamine were examined using rat aortic rings and an isolated rat lung perfusion model. Hydroxylamine and acetylcholine were equally effective in relaxing norepinephrine-contracted intact aortic rings, whereas only hydroxylamine relaxed aortic rings with endothelium removed. This endothelium-independent vasorelaxation by hydroxylamine indicated that the hydroxylamine-converting enzyme is not localized solely within endothelial cells. Catalase, an enzyme known to oxidize hydroxylamine to nitric oxide, was present in homogenates of intact and endothelium-denuded rings. Cyanamide, another catalase substrate and a known precursor of nitroxyl (HNO), was not a vasorelaxant of aortic rings or of isolated, hypoxia-constricted lungs. These results suggest that free nitroxyl is not an intermediate in the oxidation of hydroxylamine to nitric oxide. An overall pathway for the oxidative conversion of L-arginine through an hydroxylamine intermediate to nitric oxide is proposed.