Fibrinogen Saint-Germain I: a case of the heterozygous Aalpha GLY 12 --> VAL fibrinogen variant.

A fibrinogen variant was suspected based on the results of routine coagulation tests in a 2-year-old asymptomatic child. Coagulation studies showed marked prolongation of both the thrombin and reptilase times, and discrepancy was noted between the level of plasma fibrinogen as measured by a kinetic versus immunological determination. Family studies revealed that the father beared the same abnormality. Studies of purified fibrinogen revealed an impaired release of both fibrinopeptides by thrombin. Fibrin monomer polymerization and fibrin stabilization were normal. DNA sequencing revealed a heterozygous G --> T point mutation in exon 2 of the gene coding for the Aalpha chain, which substituted a Gly for Val at position 12. Although the mutation is the same as in fibrinogen Rouen, fibrinogen Saint-Germain I shows a different fibrinopeptide release pattern and a mild factor V deficiency.

Modulation of vascular tone and reactivity by nitric oxide in porcine pulmonary arteries and veins.

Isolated porcine pulmonary vessels were studied in order to evaluate the role of nitric oxide in arteries and veins. Leukotriene C4 and noradrenaline contracted porcine pulmonary arteries but induced only negligible contractions of porcine pulmonary veins. After treatment with the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (L-NOARG), significant contractions to leukotriene C4 and noradrenaline were uncovered in pulmonary veins. In arterial preparations, L-NOARG caused a less marked potentiation of noradrenaline-induced contractions and did not alter leukotriene C4-induced contractions. Endothelium-dependent relaxations to acetylcholine were greater in veins compared with arteries whereas the endothelium-independent relaxations to the nitric oxide donor sodium nitroprusside (SNP) and the cyclic nucleotide analogue 8-bromo-cGMP were similar in the two preparations. Taken together these data suggest that the apparent insensitivity of porcine pulmonary veins to leukotriene C4 and noradrenaline was because of release of nitric oxide. The effect of nitric oxide synthase inhibition was less pronounced in porcine pulmonary arteries, suggesting a preferential functional role of nitric oxide in porcine pulmonary veins, originating in a greater production of nitric oxide by veins as opposed to arteries.

Low molecular weight dextran sulfate prevents complement activation and delays hyperacute rejection in pig-to-human xenotransplantation models.

Dextran sulfate of 5000 molecular weight (DXS 5000) is known to block complement activation as well as the intrinsic coagulation cascade by potentiation of C inhibitor. The effect of DXS 5000 on hyperacute rejection (HAR) was tested in pig-to-human xenotransplantation models. For in vitro testing, a cytotoxicity assay was used with the pig kidney cell line PK15 as target cells and fresh, undiluted human serum as antibody and complement source. Ex vivo pig lung perfusion was chosen to assess DXS 5000 in a physiologic model. Pig lungs were perfused with fresh, citrate-anticoagulated whole human blood to which 1 or 2 mg/ml DXS 5000 were added; the lungs were ventilated and the blood de-oxygenated. Pulmonary vascular resistance (PVR) and blood oxygenation (deltapO2) were monitored throughout the experiment. Autologous pig blood and human blood without DXS 5000 served as controls. In the PK 15 assay DXS 5000 led to a complete, dose-dependent inhibition of human serum cytotoxicity with an average IC50 of 43 +/- 18 microg/ml (n=8). Pig lungs perfused with untreated human blood (n=2) underwent HAR within 105 +/- 64 min, characterized by increased PVR, decrease of deltapO2, and generalized edema. Microscopically, capillary bleeding as well as deposition of human antibodies, complement and fibrin could be observed. Addition of DXS 5000 (n=4) prolonged lung survival to 170 +/- 14 min for 1 mg/ml and 250 +/- 42 min for 2 mg/ml. and PVR values as well as edema formation were comparable to control lungs that were perfused with autologous pig blood (n=2). Activation of complement (activation products in serum, deposition on lung tissue) and the coagulation system (fibrin monomers) were significantly diminished as compared to human blood without DXS 5000. Binding of anti-Gal antibodies was not influenced, and in vitro experiments showed no evidence of complement depletion by DXS 5000. In conclusion, DXS 5000 is an efficient complement inhibitor in pig-to-human xenotransplantation models and therefore a candidate for complement-inhibitory/anti-inflammatory therapy either alone or in combination with other substances and warrants further investigation.

Changes in venous return parameters associated with univentricular Fontan circulations.

To clarify the physiology of venous return (Q(vr)) in Fontan circulations, venous return conductance (G(vr)) and mean circulatory filling pressure (P(mcf)) were determined in pentobarbital sodium-anesthetized pigs. Relationships between Q(vr) and right (biventricular, n = 8) or left (Fontan, n = 8) filling pressures are described by straight lines with significant correlation coefficients. Estimated P(mcf) values were correlated with observed P(mcf) values in either circulations (P

Endothelial nitric oxide synthase function in pig lung after chronic pulmonary artery obstruction.

Because long-term pulmonary artery (PA) obstruction is associated with expansion of the systemic blood supply to the lung, chronic ischemia may not occur, and endothelium nitric oxide synthase (eNOS) function may be preserved in postobstructive pulmonary arteries. To test this hypothesis, we studied piglets 2 d or 5 wk after left PA ligation or a sham operation. We measured left lung ATP and lactate lung concentrations; calcium-dependent and calcium-independent NOS activities and eNOS protein; and left PA relaxations in response to acetylcholine, calcium ionophore, and sodium nitroprusside. Decreases in ATP and increases in lactate concentrations were significantly attenuated after 5 wk PA occlusion (p < 0.05 versus sham and 2-d ligation). Compared with sham and 2-d PA occlusion, calcium-dependent NOS activity and eNOS protein were lower in the long-term PA occlusion group. Calcium-independent NOS activity was unchanged. Acetylcholine and calcium ionophore relaxations were impaired after 5 wk, whereas only acetylcholine relaxation was impaired after 2-d PA occlusion. Relaxation to sodium nitroprusside remained unchanged. In conclusion, despite relative conservation of lung energy metabolism, prolonged PA occlusion decreased eNOS function and protein in postobstructive pulmonary arteries.

Antagonist resistant contractions of the porcine pulmonary artery by cysteinyl-leukotrienes.

The contractile response to cysteinyl-leukotrienes was studied in isolated porcine pulmonary arterial rings. In endothelium-denuded preparations, the concentration-response curves for leukotriene C(4) and leukotriene D(4) were identical, whereas leukotriene E(4) did not contract these tissues. The response to leukotriene C(4) was not blocked by either CysLT(1)/CysLT(2) receptor antagonism or by pre-treatment with leukotriene E(4). In preparations with an intact endothelium, leukotriene C(4) was somewhat more potent than leukotriene D(4) and the concentration-response curves were only slightly depressed in the presence of either ICI 204,219 (4-(5-cyclopentyloxycarbonylamino-1-methylindol-3-ylmethy l)-3-methoxy -N-o-tolylsulfonylbenzamide, 1 microM) or BAY u9773 (6(R)-(4'-carboxyphenylthio)-5(S)-hydroxy-7(E),9(E), 11(Z)14(Z)-eicosatetrenoic acid, 3 microM). Indomethacin (1.7 microM) significantly reduced the response to leukotriene C(4) whereas the response to leukotriene D(4) was unchanged. These findings suggest that a CysLT receptor subtype resistant to current antagonists mediated the major part of the contractions to leukotriene C(4) and leukotriene D(4) in intact preparations, and was the sole receptor associated with contractions of endothelium-denuded preparations.

Left ventricular alterations in a model of fetal left ventricular overload.

Congenital aortic coarctation is well tolerated by the fetus because the foramen ovale and ductus arteriosus equalize intracardiac and great arteries pressures and shunts. The pathologic consequences only emerge after birth with closure of the foramen ovale and ductus arteriosus. There is, however, no documentation of myocardial effects in utero of the left ventricular (LV) pressure overload induced by aortic banding. We investigated whether prenatal aortic banding could be detrimental at the structural and/or functional level. The goal of the present study was to investigate the cardiac effects of LV pressure overload in a fetal lamb model. Nine fetal lambs underwent preductal banding of the aortic arch in utero at midgestation (CoA group), whereas their twins underwent sham surgery. All fetuses were studied between 27 and 37 d after surgery for LV pressure, anatomic and histologic anomalies, and steady state sarcoendoplasmic reticulum calcium ATPase (SERCA 2a) mRNA and protein levels and pump activity. Surgery resulted in severe aortic coarctation in all the animals in the CoA group and was associated with a 65% increase in the LV weight to body weight ratio relative to the sham-operated group (p < 0.001). Hemodynamic and histologic studies showed an evolutionary pattern depending on duration of the experimental coarctation with a shift occurring at 30 d of coarctation. The initial response of cardiomyocytes to ventricular overload was hypertrophy of the myocytes, followed by myocyte hyperplasia. Compared with sham, there was an apparent decrease in the percentage of binucleated cells in the CoA group after 30 d of coarctation. The earliest response to LV pressure overload appears to occur at the molecular level. Indeed, sarcoendoplasmic reticulum calcium ATPase (SERCA 2a) mRNA levels fell significantly to only 28.6% of the sham group value (p = 0.023), independently of the duration of coarctation. In the fetal lamb, the pressure overload-induced hypertrophy resulting from progressive aortic coarctation leads to hemodynamic and lesional abnormalities and slows ontogenic maturation.

In vivo regulation of von willebrand factor synthesis: von Willebrand factor production in endothelial cells after lung transplantation between normal pigs and von Willebrand factor-deficient pigs.

To evaluate the regulation of plasma von Willebrand factor (vWF) and its in situ production by endothelial cells (ECs), 12 swine leukocyte antigen (SLA)-compatible left lung transplantations were performed. Normal lungs were transplanted into 10 pigs homozygous for von Willebrand disease and into 2 normal pigs. Additionally, 1 normal pig underwent pneumonectomy, and 1 SLA-incompatible lung transplantation between normal pigs was performed. None of the transplanted animals received immunosuppressive therapy. Plasma vWF level was evaluated by ELISA and multimeric pattern. EC vWF content was assessed by immunohistochemistry. Global hemostasis was assessed by standardized ear bleeding time. Six of 12 SLA-compatible lung transplantations and the incompatible transplantation were successful and were used for the study. The functions and the viability of ECs, reflected by their ability to produce vWF and normal multimeric plasma vWF pattern, were preserved in SLA-compatible and -incompatible lung transplantations. vWF production was preserved in ECs that initially synthesized it. EC constitutive and storage pathways are modulated differently according to transplantation compatibility and severity of rejection. In SLA-compatible lung transplantations without histological evidence of rejection, the production of vWF was preserved, whereas constitutive vWF secretion appeared to be altered in cases with minor histological signs of rejection. In pigs with von Willebrand disease that were transplanted with normal lungs without sign of rejection, plasma vWF was significantly increased in an amount expected from the estimated production of a normal lung. In the transplanted normal lung, there was no vWF overexpression by the ECs and no recruitment of ECs that initially did not express vWF. In SLA-incompatible transplantation, ECs were morphologically normal with increased and blurred vWF labeling, whereas plasma vWF levels remained normal, reflecting that EC activation is associated with an increased vWF production with probable diversion to storage pathway. This model depicts the changes of EC regulation of vWF secretion in pig lung transplants. However, this model cannot be directly extrapolated to human organ transplantation because animals did not receive any immunosuppressive therapy, which may be toxic to ECs.

Vascular endothelium viability and function after total cardiopulmonary bypass in neonatal piglets.

Endothelium dysfunction with severe pulmonary hypertension may occur after total cardiopulmonary bypass (CPB) in infants as a result of a widespread inflammatory response. The aim of this study was to separate out the effects of lung ischemia-reperfusion from membrane oxygenator-induced activation of leukocytes on the function and viability of the pulmonary and systemic endothelia in neonatal piglets submitted to 90-min total CPB followed by 60-min reperfusion or in sham animals. Hemodynamics, gas exchange, endothelial-dependent relaxation in pulmonary and femoral arteries, and lung and skeletal muscle myeloperoxidase activity were assessed before, during, and after CPB, i.e., after reperfusion. Pulmonary and aortic endothelial cells and circulating leukocytes were harvested to assess reperfusion-induced changes in endothelial cells' viability and proliferation, and leukocyte-endothelial cell adhesion and cytotoxicity. Gas exchange worsened after reperfusion with pulmonary hypertension, increase in lung but not skeletal myeloperoxidase, and reduction of endothelial-dependent relaxation in pulmonary but not femoral arteries. After reperfusion, viabilities of pulmonary and aortic endothelial cells were reduced to 50%, endothelial cell growths were faster in pulmonary arteries than aorta, and leukocyte-pulmonary endothelial cell adhesion and cytotoxicity increased. These results suggest that in total CPB lung ischemia-reperfusion aggravates the inflammatory response and predisposes the lung endothelium to leukocyte-mediated injury.

Lung reperfusion injury after chronic or acute unilateral pulmonary artery occlusion.

Because the lungs receive their blood supply from both the pulmonary and bronchial systems, chronic pulmonary artery obstruction does not necessarily result in severe ischemia. Ischemia-reperfusion (IR) lung injury may therefore be attenuated after long-term pulmonary artery obstruction. To test this hypothesis, isolated left lungs of pigs were reperfused two days (acute IR group) or 5 wk (chronic IR group) after left pulmonary artery ligation and compared to those of sham-operated animals. The severity of IR-lung injury after 60 min ex vivo reperfusion of the left lung was assessed based on lung histology and measurements of filtration coefficient (Kfc), pulmonary arterial resistance (Rpa), and lung myeloperoxidase (MPO) activity. Marked bronchial circulation hypertrophy was seen in the chronic IR group. Hemorrhagic alveolar edema was found in all acute IR lungs but not in sham or chronic IR lungs. Compared with the sham-operated controls, Kfc and Rpa increased two-fold and threefold, and MPO 1.5-fold and twofold in the chronic and acute IR groups, respectively. In conclusion, IR-induced lung injury was markedly reduced when it occurred 5 wk after pulmonary artery ligation, probably because the systemic blood supply to the lung had time to develop, limiting ischemia.

Ex vivo lung model of pig-to-human hyperacute xenograft rejection.

OBJECTIVE: Our objective was to study lung hyperacute rejection in the pig-to-human xenotransplantation combination. METHODS: Pig lungs were harvested and continuously ventilated and perfused ex vivo, using a neonatal oxygenating system, with either xenogeneic unmodified human blood (n = 6) or autogeneic pig blood (n = 6). RESULTS: Autoperfused lungs displayed normal hemodynamics, oxygen extraction (arteriovenous oxygen difference), and histologic characteristics throughout the 3-hour study period. By contrast, xenoperfused lungs displayed, within 30 minutes, severe pulmonary hypertension and abolishment of arteriovenous oxygen difference culminating in massive pulmonary edema, hemorrhage, and lung failure after 115 +/- 44.2 minutes of reperfusion. Within 30 minutes, the human blood showed a significant drop of anti-alpha Gal immunoglobulin M and G xenoreactive antibodies (enzyme-linked immunosorbent assay) and complement activity, consumption of clotting factors, and hemolysis; total circulating human immunoglobulins remained substantially normal. Histologically, lungs perfused with human blood were congestive and showed alveolar edema and hemorrhage and multiple fibrin and platelet thrombi obstructing the small pulmonary vessels (arterioles, capillaries, and venules) but not large (segmental or lobar) pulmonary vessels. On immunohistologic examination, deposits of human immunoglobulin M and complement (C1q and C3) proteins were observed on the alveolar capillaries. CONCLUSIONS: This pig-to-human xenograft model suggests that the pig lung perfused with human blood has an early and violent hyperacute rejection that results in irreversible pulmonary dysfunction and failure within approximately 150 minutes of reperfusion.

Effects of various timings and concentrations of inhaled nitric oxide in lung ischemia-reperfusion. The Paris-Sud University Lung Transplantation Group.

Experimental studies reveal that inhaled nitric oxide (NO) can prevent, worsen, or have no effect on lung injury in the setting of ischemia-reperfusion (I-R). We tested the hypothesis that these disparate effects could be related to differences in the timing of administration and/or concentration of inhaled NO during I-R. Isolated rat lungs were subjected to 1-h periods of ischemia followed by 1-h periods of blood reperfusion. We investigated the effects of NO (30 ppm) given during ischemia, NO (30 or 80 ppm) begun immediately at reperfusion, or NO (30 ppm) given 15 min after the beginning of reperfusion, on total pulmonary vascular resistance (PVR), the coefficient of filtration (Kfc), the lung wet/dry weight ratio (W/D) of lung tissue, and lung myeloperoxidase activity (MPO). A control group did not receive NO. NO given during ischemia had no effect on Kfc or MPO, but decreased PVR. NO (30 ppm) during reperfusion (early or delayed) decreased PVR, W/D, Kfc and MPO. NO at 80 ppm decreased PVR and MPO but not W/D or Kfc. In conclusion, NO at 30 ppm, given immediately or in a delayed fashion during reperfusion, attenuates I-R-induced lung injury. NO at 30 ppm given during ischemia or at 80 ppm during reperfusion is not protective.

Inhaled nitric oxide and pentoxifylline in rat lung transplantation from non-heart-beating donors. The Paris-Sud University Lung Transplantation Group.

BACKGROUND: In non-heart-beating donor lung transplantation, postmortem warm ischemia poses a special challenge. Inhaled nitric oxide and pentoxifylline have been shown to attenuate ischemia-reperfusion injury after lung transplantation. We hypothesized that concomitant administration of inhaled nitric oxide and pentoxifylline would result in a synergistic effect on ischemia-reperfusion lung injury. METHODS: Lungs were harvested from non-heart-beating donors after 30 minutes of in situ warm ischemia, flushed, and stored for 2 hours at 4 degrees C before left lung transplantation in rats. Inhaled nitric oxide (30 ppm) was added during cadaver ventilation and reperfusion; pentoxifylline was given intravenously throughout reperfusion. The following groups were studied (n = 8 each): control, pentoxifylline, nitric oxide, and nitric oxide+pentoxifylline. Hemodynamic indices and arterial blood gases were obtained after ligation of the right pulmonary artery. Lung myeloperoxidase and wet/dry ratio were measured after death. RESULTS: All rats that did not receive nitric oxide died within 10 minutes after ligation. Inhaled nitric oxide significantly decreased pulmonary vascular resistance and improved recipient survival. Nitric oxide + pentoxifylline improved pulmonary vascular resistance, arterial oxygen tension, and survival even further and reduced lung myeloperoxidase as compared with the group that received nitric oxide only. Preservation solution flush time was significantly decreased in both groups receiving nitric oxide, suggesting that inhaled nitric oxide used during cadaver ventilation allows for a more even distribution of the preservation solution. CONCLUSIONS: We conclude that treatment with inhaled nitric oxide + pentoxifylline results in a synergistic protection from ischemia-reperfusion injury after non-heart-beating donor lung transplantation. This is likely the result of a dual action on the graft vasculature and neutrophil sequestration.

Inhaled nitric oxide attenuates reperfusion injury in non-heartbeating-donor lung transplantation. Paris-Sud University Lung Transplantation Group.

BACKGROUND: Non-heartbeating-donor (NHBD) lung transplantation could help reduce the current organ shortage. Polymorphonuclear neutrophil (PMN) activation plays a pivotal role in ischemia-reperfusion injury (I-R), and can be inhibited by nitric oxide (NO). We hypothesized that inhaled NO might be beneficial in NHBD lung transplantation. METHODS: The effect of inhaled NO on PMNs was studied by measuring in vivo PMN lung sequestration (myeloperoxidase activity) and adhesion of recipient circulating PMNs to cultured pulmonary artery endothelial cells (PAECs) in vitro. Pigs were randomly assigned to an NO or a control group (n=9 each). In the NO group, cadavers and recipients were ventilated with oxygen and 30 parts per million of NO. After 3 hr of postmortem in situ warm ischemia and 2 hr of cold ischemia, left allotransplantation was performed. The right pulmonary artery was ligated, and hemodynamic and gas exchange data were recorded hourly for 9 hr. Recipient PMN adherence to tumor necrosis factor-alpha- and calcium ionophore-stimulated PAECs was measured before and after reperfusion, and lung PMN sequestration was determined after death. RESULTS: NO-treated animals exhibited lowered pulmonary vascular resistance (P<0.01), as well as improved oxygenation (P<0.01) and survival (P<0.05). Adhesion of PMNs to PAECs was inhibited in the NO group before (P<0.001) and after reperfusion (P<0.0001). Lung PMN sequestration was reduced by NO (P<0.05). CONCLUSIONS: Inhaled NO attenuates I-R injury after NHBD lung transplantation. This is likely due to the prevention of I-R-induced pulmonary vasoconstriction and to the direct effect on peripheral blood PMN adhesion to endothelium, which results in reduced sequestration and tissue injury.

Prevention of ischemia-reperfusion lung injury by sulfated Lewis(a) pentasaccharide. The Paris-Sud University Lung Transplantation Group.

Inhibition of polymorphonuclear neutrophil (PMN) adhesion to the pulmonary endothelium attenuates ischemia-reperfusion (I/R) lung injury. We hypothesized that 3'-sulfated Lewis(a) (SuLa), a potent ligand for the selectin adhesion molecules, may have a beneficial effect on I/R lung injury, as measured by the filtration coefficient (K(fc)), and reduce pulmonary sequestration of PMN as assessed by the lung myeloperoxidase (MPO) activity. Blood-perfused rat lungs were subjected to 30 min of perfusion, 60 min of warm ischemia, and 90 min of reperfusion after treatment with either SuLa (200 microg) or saline. Effects of SuLa on PMN adhesion to cultured human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor-alpha and calcium ionophore were also investigated. Compared with preischemia conditions, I/R induced a significant increase in K(fc), which was attenuated with SuLa (80 +/- 8 vs. 30 +/- 5%; P < 0.001). SuLa reduced lung MPO and PMN adhesion to stimulated HUVEC. These results indicate that SuLa reduces I/R-induced lung injury and PMN accumulation in lung. This protective effect might be related to inhibition of PMN adhesion to endothelial cells.

Tracheal growth after slide tracheoplasty.

OBJECTIVE: Our goal was to investigate the effects of slide tracheoplasty on tracheal growth in newborn piglets. METHODS: Slide tracheoplasty was performed on normal trachea (n = 6) and a model of tracheal stenosis resembling that seen in infants (n = 6). After division of the trachea at its midportion between the second cartilaginous ring above and the right upper lobe takeoff below (around 23 rings), the proximal and distal segments were incised vertically on opposite anterior and posterior surfaces and reconstructed together. RESULTS: The reconstructed tracheas lengthened and their cross-sectional areas enlarged linearly at a rate of 0.94 cm per month and 1.55 mm2/kg, respectively, as the piglets grew over a 6-month period from 4.7 +/- 0.6 to 64.4 +/- 5.7 kg (+/- standard deviation). Growth was not different between the two studied groups. There was no narrowing or late restenosis. The mean anastomotic cross-sectional area was overall 1.63 +/- 0.28 times larger (range 1.2 to 2.7) than the cross-sectional area of the unreconstructed trachea. When the animals were put to death, all tracheal lumina were completely lined with normal respiratory epithelium and all layers were histologically intact; anastomotic trachealis muscles contracted less (p < 0.001) but relaxed similarly to those muscles lining normal tracheas. Tracheal blood supply was macroscopically and microscopically normal in both groups; however, newborn piglets had an almost twofold increased number of intramural capillary vessels as opposed to adult pigs (p < 0.001). CONCLUSIONS: Results suggest that slide tracheoplasty is not limited by the length of stenosis, provides a permanent enlargement of the cross-sectional airway diameter, does not compromise tracheal vascular supply, and does not impair tracheal growth as somatic growth continues.

Cholinesterase activity in pig airways and epithelial cells.

Acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) activities were detected in bronchial and bronchial epithelial cell homogenates of the pig. In the bronchial homogenates, the maximal upstroke velocity (Vmax) of AChE and the maximal velocity after second substrate fixation (Vss) of BChE were 5.70 +/- 0.46 and 7.87 +/- 0.81 mU/mg protein, respectively. In the epithelial cell homogenates, a smaller amount of cholinesterase (ChE) was found: Vmax was 0.62 +/- 0.29 and Vss was 1.56 +/- 0.33 mU/mg protein for AChE and BChE, respectively. AChE activity was increased by 21 +/- 5% in the bronchial homogenates and by 54 +/- 14% in the epithelial cell homogenates, when intact bronchial segments were incubated with a cyclooxygenase inhibitor, indomethacin (INDO). These results suggest that prostanoids may be involved in the regulation of AChE activity in pig airways.

Prevention of reperfusion injury by inhaled nitric oxide in lungs harvested from non-heart-beating donors. Paris-Sud University Lung Transplantation Group.

BACKGROUND: In lung transplantation using non-heart-beating donors (NHBD), the postmortem period of warm ischemia exacerbates lung ischemia-reperfusion injury. We hypothesized that inhaled nitric oxide (NO) would reduce ischemia-reperfusion injury, and thus ameliorate the viability of the lung graft. METHODS: A blood-perfused, isolated rat lung model was used. Lungs were flushed and harvested from non-heart-beating donors after 30 minutes of in situ warm ischemia. The lung was then stored for 2 hours at 4 degrees C. Inhaled NO at 30 ppm was given either during the period of warm ischemia, during reperfusion, or during both periods. Lung ischemia-reperfusion injury was assessed after 1 hour of reperfusion by measuring pulmonary vascular resistance, coefficient of filtration, wet-to-dry lung weight ratio, and myeloperoxidase activity. RESULTS: A severe IR injury occurred in lungs undergoing ischemia and reperfusion without NO as evidenced by high values of pulmonary vascular resistance (6.83 +/- 0.36 mm Hg. mL-1.min-1), coefficient of filtration (3.02 +/- 0.35 mL.min-1.cm H2O-1 x 100 g-1), and wet-to-dry lung weight ratio (8.07 +/- 0.45). Lower values (respectively, 3.31 +/- 0.44 mm Hg.mL-1.min-1, 1.49 +/- 0.34 mL.min-1.cm H2O-1 x 100 g-1, and 7.44 +/- 0.43) were observed when lungs were ventilated with NO during ischemia. Lung function was further improved when NO was given during reperfusion only. All measured variables, including myeloperoxidase activity were significantly improved when NO was given during both ischemia and reperfusion. Myeloperoxidase activity was significantly correlated with coefficient of filtration (r = 0.465; p < 0.05). CONCLUSIONS: These data suggest that inhaled NO significantly reduces ischemia-reperfusion injury in lungs harvested from non-heart-beating donors. This effect might be mediated by inhibition of neutrophil sequestration in the reperfused lung.

Lasting beneficial effect of short-term inhaled nitric oxide on graft function after lung transplantation. Paris-Sud University Lung Transplantation Group.

The combination of ischemia and reperfusion after lung transplantation is characterized by endothelial damage, neutrophil sequestration, and decreased release of endothelial nitric oxide. Because nitric oxide has been shown to selectively dilate the pulmonary vasculature, abrogate neutrophil adherence, and restore endothelial dysfunction, we hypothesized that inhaled nitric oxide given for 4 hours during initial reperfusion might attenuate reperfusion injury in a porcine model of left single-lung transplantation. We tested hemodynamic and gas exchange data, lung neutrophil sequestration, and pulmonary artery endothelial dysfunction after 4 and 24 hours of reperfusion in 12 pigs randomly assigned to nitric oxide and control groups. Harvested lungs were preserved in normal saline solution for 24 hours at 4 degrees C. During transplantation, inflatable cuffs were placed around each pulmonary artery to allow separate evaluation of each lung by occluding flow. Compared with the transplanted lungs in the control group, transplanted lungs in pigs treated with inhaled nitric oxide significantly improved gas exchange, pulmonary vascular resistance, shunt fraction, and oxygen delivery at 4 and 24 hours after reperfusion. Neutrophil sequestration, as measured by the neutrophil-specific enzyme myeloperoxidase and the alveolar leukocyte count per light microscopic field, was significantly lower at 24 hours after reperfusion in the transplanted lungs of the nitric oxide group. The nitric oxide-treated native right lungs exhibited significantly reduced increase in neutrophil accumulation compared with that in control native right lungs. After 24 hours of reperfusion, endothelium-dependent relaxation to acetylcholine was similarly and severely altered in both groups. We conclude that short-term inhaled nitric oxide given during the first 4 hours of reperfusion after lung transplantation significantly attenuates reperfusion injury, improving graft function as long as 24 hours after operation. This effect is probably mediated by a decrease in neutrophil sequestration. A protective effect on the contralateral lung was also observed. Inhaled nitric oxide may be a suitable agent when an acute reperfusion phenomenon is anticipated.