Guy-Michel Mazmanian

Lasting beneficial effect of short-term inhaled nitric oxide on graft function after lung transplantation☆☆☆★★★♢

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.

Experimental tracheal and tracheoesophageal allotransplantation

We investigated the effects of allograft perfusion with a preservative technique and of combined thyrotracheoesophageal implantation on airway epithelium of long segments of thyrotracheal grafts allotransplanted on their own vascular pedicles into immunosuppressed pigs. Four groups of five animals each underwent heterotopic (into the neck) thyrotracheal (group 1) and thyrotracheoesophageal (group 2) and orthotopic thyrotracheal (group 3) and thyrotracheoesophageal (group 4) allotransplantation. Allograft revascularization included (1) interposition of donor right subclavian artery—incorporating the inferior thyroid artery—to recipient right carotid artery (end-to-end fashion) and (2) end-to-side anastomosis of donor anterior vena cava to recipient right external jugular vein. All thyrotracheoesophageal blocks were harvested after inferior thyroid artery perfusion with 4° C Euro-Collins solution. The overall lengths of tracheal and esophageal grafts were 10.7 ± 2.7 cm and 13.4 ± 3.6 cm, respectively. In the heterotopic groups, all allografts were viable and histologically normal at postmortem examination and the incidence and severity of airway ischemia and rejections (at equal residual levels of cyclosponine) were not different between groups 1 and 2. In the orthotopic groups, the first two pigs died of airway collapse with histologically normal grafts. In the remaining pigs, temporary airway stenting was inserted and allografts remained viable and histologically intact for their entire length 30 days after transplantation. Transplanted tracheal smooth muscles had concentration-dependent contractions and relaxations similar to those of nontransplanted (native) tracheas. This study documents the feasibility of allotransplanting long tracheal and esophageal segments on their own vascular pedicles and demonstrates that allograft preservation and thyrotracheoesophageal transplantation are equally effective in minimizing airway ischemia. Thyrotracheoesophageal transplantation does not enhance recipient alloimmune response compared with thyrotracheal transplantation alone.

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.

Heterotopic pig model for direct revascularization and venous drainage of tracheal allografts

A macrosurgical technique of thyrotracheal harvesting and direct revascularization with and without venous drainage in a heterotopic thyrotracheal and immunosuppressed allograft in the pig model is described. Harvesting included en bloc cervicothoracic exenteration of the aortic arch and its supraortic trunks, anterior vena cava, jugular veins, subclavian vessels, thyroid gland, cervicothoracic trachea, and esophagus. This technique conserves the tracheal arterial supply provided by either the right or left subclavian artery, directly or indirectly via the inferior thyroid artery, and venous return provided by the anterior vena cava, directly or indirectly via the descending cervical vein. In recipients, implantation included (1) arterial end-to-end anastomoses of the proximal and postscalenic stumps of donors subclavian artery to the proximal and prescalenic stumps of recipients subclavian artery; (2) end-to-side venous anastomosis of the donors anterior vena cava to the recipients brachiocephalic venous trunk; and (3) heterotopic implantation of the proximal and distal orifices of the grafted trachea into the neck. Ten adult Large White pigs underwent direct revascularization of a thyrotracheal allograft with (n = 6, group 1) and without (n = 4, group 2) venous drainage. All grafts of group 2 exhibited a venous infarction, extensive inferior thyroid artery thrombosis, and ischemic and suppurative thyrotracheal necrosis 1 to 2 days after transplantation. In group 1, the length of the grafted trachea and number of rings were 9.75 +/- 1.5 cm and 22.1 +/- 3.3, respectively; ischemic time was 236.3 +/- 338.3 minutes. Group 1 pigs were put to death 4 (n = 4) and 3 (n =2) weeks after transplantation. All tracheal grafts had histologically normal airway epithelium; isolated areas of necrotic ischemia of the chorion and submucosa lasted for the first 7 days after transplantation but disappeared after epithelial regeneration. Premortem angiograms showed that all vascular anastomoses were patent. Grafts were histologically normal at postmortem examinations and all but one had no rejection. This large animal model demonstrates that long tracheal allografts might be transplanted by means of this direct revascularization and venous drainage technique.

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 Reperfusion Injury by Inhaled Nitric Oxide in Lungs Harvested From Non-Heart-Beating Donors

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.

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.

l-arginine and pentoxifylline attenuate endothelial dysfunction after lung reperfusion injury in the rabbit

BACKGROUND Among the factors involved in the early complications of lung transplantation is the ischemia-reperfusion syndrome related to a warm reperfusion in ischemic lungs. METHODS Using an isolated rabbit lung preparation perfused with whole blood, we studied the effects of cold ischemia followed by a warm reperfusion on pulmonary vascular responses to reproduce experimentally the conditions encountered during lung transplantation. RESULTS Pulmonary vascular responses to acetylcholine were rapidly altered by warm ischemia (relaxation of 7% versus 60% in controls). Conversely, relaxation was maintained even after a prolonged cold ischemic storage (maximal relaxation of 57% at 48 hours). Warm reperfusion in ischemic lungs induced major alteration of endothelium-dependent relaxation (maximal relaxation of 13% at 4 hours). The addition of L-arginine or pentoxifylline during reperfusion prevented the pulmonary endothelial alteration resulting from warm reperfusion. CONCLUSION These data suggest that treatments aimed at maintaining intact functional endothelium reduce ischemia-reperfusion injury in transplanted lungs.

Inhaled nitric oxide and pentoxifylline in rat lung transplantation from non-heart-beating donors

Abstract 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° C before left lung transplantation in rats. Inhaled nitric oxide (30 ppm) was added during cadaver ventilation and reperfusion; pentoxifylline was given intravenously throughout reperfrsion. 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. Conclusion: 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. (J Thorac Cardiovasc Surg 1997;113:821-9)

Maximal preservation time of tracheal allografts

Background. Our objective was to study the maximal preservation time of directly revascularized tracheal allografts in immunosuppressed piglets. Methods. Donor grafts were flushed with Euro-Collins solution (65 mL/kg at 4°C) by simultaneous inferior thyroid artery and bronchial artery perfusion through a 15-cm aortic segment and heterotopically implanted on their own vascular pedicle after 3 (group 1), 6 (group 2), 15 (group 3), and 24 (group 4) hours of static storage in Euro-Collins solution at 4°C (n = 5 each). The animals were observed for 4 weeks after transplantation and then sacrificed. Histologic evaluation of the tracheal allografts was routinely done using specimens from open biopsies. Results. The overall length of tracheal grafts was 12.4 ± 0.6 cm, and this variable was not significantly different between the four groups. Graft exocrine (mucous secretion) function began 1.3 ± 0.5 days after transplantation in groups 1 through 3 but was absent in all group 4 grafts ( p p p Conclusions. These results demonstrate that tracheal allografts may be safely preserved for as long as 15 hours and that longer periods of ischemia are likely to result in irreversible allograft damage.