Novel critical role of Toll-like receptor 4 in lung ischemia-reperfusion injury and edema.

Toll-like receptors (TLRs) of the innate immune system contribute to noninfectious inflammatory processes. We employed a murine model of hilar clamping (1 h) with reperfusion times between 15 min and 3 h in TLR4-sufficient (C3H/OuJ) and TLR4-deficient (C3H/HeJ) anesthetized mice with additional studies in chimeric and myeloid differentiation factor 88 (MyD88)- and TLR4-deficient mice to determine the role of TLR4 in lung ischemia-reperfusion injury. Human pulmonary microvascular endothelial monolayers were subjected to simulated warm ischemia and reperfusion with and without CRX-526, a competitive TLR4 inhibitor. Functional TLR4 solely on pulmonary parenchymal cells, not bone marrow-derived cells, mediates early lung edema following ischemia-reperfusion independent of MyD88. Activation of MAPKs and NF-kappaB was significantly blunted and/or delayed in lungs of TLR4-deficient mice as a consequence of ischemia-reperfusion injury, but edema development appeared to be independent of activation of these signaling pathways. Pretreatment with a competitive TLR4 inhibitor prevented edema in vivo and reduced actin cytoskeletal rearrangement and gap formation in pulmonary microvascular endothelial monolayers subjected to simulated warm ischemia and reperfusion. In addition to its well-accepted role to alter gene transcription, functioning TLR4 on pulmonary parenchymal cells plays a key role in very early and profound pulmonary edema in murine lung ischemia-reperfusion injury. This may be due to a novel mechanism: regulation of endothelial cell cytoskeleton affecting microvascular endothelial cell permeability.

Ex-vivo perfusion and ventilation of rat lungs from non-heart-beating donors before transplant.

BACKGROUND: We developed an ex-vivo circuit to evaluate human lungs retrieved from non-heart-beating donors. We assessed the effect of a similar circuit on the function of transplanted rat lungs retrieved from non-heart-beating donors. METHODS: One hour after death, Sprague-Dawley rat heart-lung blocks were flushed with 20 mL of cold Perfadex, stored cold for 1 hour, then warmed to 37 degrees C in a circuit perfused with Earle's crystalloid solution with or without washed porcine erythrocytes (hematocrit 12% to 15%). At 37 degrees C, lungs were ventilated for 15 minutes with alveolar gas, perfusion-cooled to 20 degrees C, flushed again with cold Perfadex, and then stored cold for 2.5 hours. The left lung was transplanted using a modified cuff technique with flow probes on the main and left pulmonary arteries. After 1 hour of reperfusion, arterial blood gases from the left pulmonary vein and wet/dry weight ratio (W/D) of both donor lungs were determined. Lungs transplanted after retrieval from heart-beating or non-heart-beating donors served as controls (n = 6 per group). RESULTS: Lungs gained weight in the circuit but W/D and PO2 were similar after transplantation for all groups. After transplantation, vascular resistance was higher and dynamic compliance was lower for lungs perfused in the circuit. Myeloperoxidase and conjugated diene levels were modestly elevated in lungs transplanted from non-heart-beating donors irrespective of perfusion in the circuit. CONCLUSIONS: Rat lungs are suitable for transplant after ex-vivo perfusion and ventilation. This model closely mimics methods used to evaluate the function of lungs retrieved from human non-heart-beating donors and can economically evaluate ex-vivo therapies for lungs retrieved from non-heart-beating donors.

Inhaled nitric oxide reduces ischemia-reperfusion injury in rat lungs from non-heart-beating donors.

OBJECTIVE: If lungs could be retrieved from non-heart-beating donors, the critical shortage of lungs for transplantation could be alleviated. However, lungs subjected to warm ischemia develop edema when reperfused. We hypothesized that ventilation of rat lungs from non-heart-beating donors with nitric oxide during the period of warm ischemia alone, with reperfusion, or both might reduce ischemia-reperfusion injury. METHODS: An isolated perfused rat lung model measured the filtration coefficient and accumulation of lung water by the wet/dry weight ratio. Donor rats were euthanized, and then lungs were retrieved immediately after death or 2 or 3 hours postmortem. Lungs retrieved postmortem were either not ventilated or ventilated with 100% oxygen alone or 40 ppm nitric oxide in oxygen. In the circuit, lungs were ventilated with alveolar gas with or without 40 ppm nitric oxide. RESULTS: Nitric oxide administration to the non-heart-beating donor or in the perfusion circuit reduced filtration coefficient and wet/dry weight ratio. Lungs retrieved 2 hours postmortem ventilated with nitric oxide or treated with nitric oxide on reperfusion had filtration coefficients and wet/dry weight ratios similar to those of lungs retrieved immediately after death. Nitric oxide was most beneficial when administered both during warm ischemia and at reperfusion in lungs retrieved 3 hours postmortem. Nitric oxide administration in the circuit was associated with increased lung levels of lung cyclic guanosine monophosphate, determined by enzyme-linked immunosorbent assay. CONCLUSIONS: Administration of nitric oxide to non-heart-beating donors during warm ischemia and with reperfusion might facilitate transplantation of lungs from non-heart-beating donors by reducing ischemia-reperfusion injury and capillary leak.

Ex vivo evaluation of human lungs for transplant suitability.

BACKGROUND: If lungs could be retrieved from non-heart-beating donors, the critical shortage of lungs for transplant could be alleviated. An obstacle to this approach is the inability to predict these lungs' suitability for transplant. We used human lungs deemed unsuitable for transplant to develop a method to perfuse and ventilate human lungs ex vivo to assess gas exchange and vascular resistance, and to perform bronchoscopic inspection and radiographic evaluation. METHODS: Lungs were retrieved from six brain-dead organ donors after cold Perfadex (Vitrolife, Kungsbacka, Sweden) flush, stored cold for 6 to 13 hours (mean, 8.7 hours) then perfused and rewarmed in a modified cardiopulmonary bypass circuit. Circuit perfusate was buffered colloid-crystalloid containing type-specific leukocyte-filtered blood (hematocrit of 10%-12%), circulated through a membrane oxygenator ventilated with CO2 and nitrogen to deoxygenate it. Lungs were ventilated with fraction of inspired oxygen (Fio2) 0.5 when 32 degrees C was reached. Gas exchange and vascular resistance was assessed at 5 L/minute flow at 37 degrees C, Fio2 0.5 and 1.0. Bronchoscopy, plain radiographs, and spiral computed tomographic (CT) scans were performed. Lung biopsies were obtained pre- and post-reperfusion. RESULTS: Ex vivo perfusion did not cause increased wet to dry ratio, or major abnormalities by microscopy but was associated with elevated tissue levels of conjugated dienes. The alveolar-arterial difference in partial pressure of oxygen (Pao2)/Fio2 ratio in the ex vivo circuit was generally higher than in the six donors. Ex vivo radiographs and CT scans were abnormal in all lungs, confirming unsuitability of these lungs for transplant. CONCLUSIONS: Ex vivo evaluation of human lungs is feasible and may be useful to evaluate transplant suitability of lungs retrieved after circulatory arrest from non-heart-beating donors.

Nitroglycerin reperfusion reduces ischemia-reperfusion injury in non-heart-beating donor lungs.

BACKGROUND: Lung transplantation is severely limited by an inadequate supply of lungs from brain-dead donors. A potential solution is use of lungs from non-heart-beating donors (NHBDs) with retrieval at intervals after circulatory arrest and death. A warm ischemic period with concomitant reperfusion injury is a major limiting factor in the transplantation of lungs retrieved from NHBDs. We hypothesized that the administration of the nitric oxide-donor nitroglycerin to lungs from NHBDs would reduce ischemia-reperfusion injury by activation of guanylate cyclase to form guanosine 3',5'-cyclic monophosphate (cGMP). METHODS: An in situ isolated perfused rat lung model was used. Lungs were retrieved from rats at varying intervals after circulatory arrest and death. Lungs were either ventilated with O(2) in situ or not ventilated. Lungs were reperfused at intervals after death with Earle's solution with or without nitroglycerin (0.1 mg/ml). Lung ischemia-reperfusion injury was assessed by capillary filtration coefficient, wet-to-dry lung weight ratio, and pulmonary hemodynamics. Tissue levels of adenine nucleotides and cGMP concentrations were measured by high-performance liquid chromatography and enzyme immunoassay, respectively. RESULTS: Reperfusion with nitroglycerin decreased capillary filtration coefficient compared with reperfusion without nitroglycerin at all post-mortem ischemic times, irrespective of pre-harvest ventilation. cGMP levels increased significantly with nitroglycerin-reperfusion and attenuated decreases in high-energy adenine nucleotides. CONCLUSIONS: Reperfusion of lungs with nitroglycerin may facilitate safe lung transplantation from NHBDs by reducing capillary leak after reperfusion.

Trends in lung pH and PO2 after circulatory arrest: implications for non-heart-beating donors and cell culture models of lung ischemia-reperfusion injury.

BACKGROUND: A better understanding of lung tissue environment after circulatory arrest would allow more accurate cell culture models to study ischemia-reperfusion lung injury and facilitate retrieval of lungs from non-heart-beating donors. METHODS: To establish the time course of changes in pH and PO2 in lung tissue after circulatory arrest, 12 Sprague-Dawley rats were sacrificed. After sternotomy, pH and PO2 microelectrodes were inserted into the lungs and sealed by application of Focal Seal. Rats were maintained at normothermia (37 degrees C). Two groups of rats (n = 6 atelectatic, n = 6 room air-inflated) were followed for 4 hours after arrest, when lung tissue adenine nucleotide levels were measured by chromatography and cell death was quantified by trypan blue exclusion. Human umbilical vein endothelial cells underwent simulated ischemia and 6 hours of cold storage by replacement of culture medium with cold Perfadex. Interleukin (IL)-6 and IL-8 were measured in medium 21 hours later by enzyme-linked immunosorbent assay (ELISA). RESULTS: In both groups of rats, lung [H+] increased linearly with time. In atelectatic lungs, PO2 fell precipitously, but in inflated lungs, PO2 decreased linearly for 60 to 75 minutes post-mortem and then became stable. After 4 hours at 37 degrees C, most parenchymal lung cells were dead in both groups. IL-6 and IL-8 levels increased significantly in medium of cultured endothelial cells subjected to cold storage without hypoxia. CONCLUSIONS: In room-air-inflated lungs maintained at 37 degrees C, oxygen consumption continues for at least 1 hour after circulatory arrest. Warm atelectasis is poorly tolerated. Hypothermic storage can induce elaboration of cytokines by endothelial cells in the absence of hypoxia.

Isoproterenol reduces ischemia-reperfusion lung injury despite beta-blockade.

BACKGROUND: If lungs could be retrieved from non-heart-beating donors (NHBDs), the shortage of lungs for transplantation could be alleviated. The use of lungs from NHBDs is associated with a mandatory warm ischemic interval, which results in ischemia-reperfusion injury upon reperfusion. In an earlier study, rat lungs retrieved 2-h postmortem from NHBDs had reduced capillary leak measured by filtration coefficient (Kfc) when reperfused with isoproterenol (iso), associated with an increase in lung tissue levels of cyclic AMP (cAMP). The objective was to determine if this decrease in Kfc was because of beta-stimulation, or would persist despite beta-blockade. MATERIALS AND METHODS: Donor rats were treated intraperitoneally with beta-blockade (propranolol or pindolol) or carrier, sacrificed, and lungs were retrieved immediately or 2 h postmortem. The lungs were reperfused with or without iso and the beta-blockers in the reperfusate. Outcome measures were Kfc, wet:dry weight ratio (W/D), lung levels of adenine nucleotides and cAMP. RESULTS: Lungs retrieved immediately after death had normal Kfc and W/D. After 2 h of ischemia, Kfc and W/D were markedly elevated in controls (no drug) and lungs reperfused with beta-blockers alone. Isoproterenol-reperfusion decreased Kfc and W/D significantly (P < 0.01) even in the presence of beta-blockade. Lung cAMP levels were increased only with iso in the absence of beta-blockade. CONCLUSIONS: The attenuation of ischemia-reperfusion injury because of iso occurs even in the presence of beta-blockade, and may not be a result of beta-stimulated increased cAMP.