Evaluating lung injury at increasing time intervals in a murine brain death model.

INTRODUCTION: Only 15%-25% of brain death (BD) donors match the ideal donor criteria for lung transplantation. Lung injury may evolve in the hours after onset of brain death, but the evolution over time has not been well studied in lung. The aim of this study was to evaluate lung injury at different time points after BD using a murine model. MATERIALS AND METHODS: Male C57BL6/J mice (8-10 wk) were anesthetized, tracheotomized, and mechanically ventilated. Mice were randomly assigned to six groups (n=8/group): 1 h, 3 h, and 6 h sham ([SH1], [SH3], [SH6]) and 1 h, 3 h, and 6 h brain death ([BD1], [BD3], [BD6]). BD was gradually induced by a subdural balloon catheter. Heart rate and mean arterial pressure were continuously monitored. At the end of the experiment, bronchoalveolar lavage was performed and the left lung was excised for histopathologic analysis. RESULTS: The Cushing reflex was characterized by a rapid increase in heart rate and mean arterial pressure after balloon inflation in BD animals. An increase in percentage of neutrophils was seen with a longer follow-up period (P<0.05). Interleukin 6 and interleukin 10 levels in bronchoalveolar lavage progressively increased with longer time intervals after BD ([BD1] versus [BD6]; P<0.01). Histologic signs of lung injury (congestion, hemorrhage, and neutrophilic influx) were more pronounced in [BD3] and [BD6] compared with the other groups; however, this difference did not reach statistical significance. CONCLUSION: Three hours after brain death, significant signs of inflammation and lung injury were seen compared with sham-operated animals. This murine BD model gives us opportunities for further mechanistic studies regarding treatment of BD-related donor lung injury.

Fluoro-D-glucose-micro positron emission tomography as a diagnostic tool to confirm brain death in a murine donor lung injury model.

PURPOSE: Because brain death (BD)-related donor lung injury is still poorly understood, a reliable mouse model can help in understanding the immunologic mechanisms behind this lung injury. The purpose of our study was to validate BD in mice using small-animal positron emission tomography. PROCEDURES: BD was induced in male Balb/c mice (27.1 ± 0.9 g) with an intracranial balloon catheter inflated rapidly (<1 min) [BD](R) or gradually (36 ± 5 min) [BD](G), and compared with sham-operated [SH] and control animals [C] (n = 6/group). Ten minutes after balloon insertion 10.4 ± 1.0 MBq 2-deoxy-2-[(18)F]-fluoro-D-glucose ((18)FDG) was administered intravenously and static images were performed and quantified. RESULTS: Coronal, sagittal, and transaxial sections of cerebral (18)FDG activity revealed significant differences when comparing [BD](R) and [BD](G) with [C] and [SH] animals. No significant (18)FDG uptake was visually detectable in [BD](R) and [BD](G). The percentage injected dose showed significant differences between BD groups and [C] and [SH] (P < 0.0001). No significant difference was seen between [C] versus [SH] nor between [BD](R)versus [BD](G) (P > 0.05). CONCLUSIONS: (18)FDG micro positron emission tomography imaging is a valuable tool to demonstrate brain functionality and can therefore be used as a surrogate test to confirm BD in mice.

Chronic rejection pathology after orthotopic lung transplantation in mice: the development of a murine BOS model and its drawbacks.

Almost all animal models for chronic rejection (CR) after lung transplantation (LTx) fail to resemble the human situation. It was our attempt to develop a representative model of CR in mice. Orthotopic LTx was performed in allografts receiving daily immunosuppression with steroids and cyclosporine. Controls included isografts and mice only undergoing thoracotomy (SHAM). Allografts were sacrificed 2, 4, 6, 8, 10 or 12 weeks after LTx. Pulmonary function was measured repeatedly in the 12w allografts, isografts and SHAM mice. Histologically, all allografts demonstrated acute rejection (AR) around the blood vessels and airways two weeks after LTx. This decreased to 50-75% up to 10 weeks and was absent after 12 weeks. Obliterative bronchiolitis (OB) lesions were observed in 25-50% of the mice from 4-12 weeks. Isografts and lungs of SHAM mice were normal after 12 weeks. Pulmonary function measurements showed a decline in FEV(0.1), TLC and compliance in the allografts postoperatively (2 weeks) with a slow recovery over time. After this initial decline, lung function of allografts increased more than in isografts and SHAM mice indicating that pulmonary function measurement is not a good tool to diagnose CR in a mouse. We conclude that a true model for CR, with clear OB lesions in about one third of the animals, but without a decline in lung function, is possible. This model is an important step forward in the development of an ideal model for CR which will open new perspectives in unraveling CR pathogenesis and exploring new treatment options.

Preemptive therapy with steroids but not macrolides improves gas exchange in caustic-injured donor lungs.

BACKGROUND: Donor lungs are susceptible to aspiration in the period before and after brain death. We hypothesized that preemptive anti-inflammatory treatment may result in better graft performance as evaluated during ex vivo lung perfusion (EVLP). METHODS: Pigs (27.4 ± 8.3kg) were divided into four groups (n = 6). Group [S] was treated with steroids (methylprednisolone 10 mg/kg), group [M] with macrolides (clarithromycin 250 mg), and group [P] with placebo (saline). In all groups, treatment was given i.v. 25, 18, and 1 h before instillation of 4 mL/kg gastric juice (GJ). Group [C] without GJ instillation served as a control group. Two hours after the onset of acute lung injury, lungs were flushed, explanted, and stored cold for 75 min. Graft performance was then assessed during EVLP for 2 h. Aerodynamic and hemodynamic parameters and oxygenation capacity (∆PO(2)) were recorded every 30 min (T30-T120). BAL samples were collected and analyzed for total and differential cells, IL-6, IL-1ß, 8- isoprostane, and CRP levels. Wet-to-dry weight ratio [W/D] was measured and tissue samples were collected for histology. RESULTS: ΔPO(2) in [S] was comparable to [C] and was higher versus [M] (P < 0.05) and [P] (P < 0.01). No differences were observed in pulmonary vascular resistance, lung compliance, and mean airway pressure. No additional edema was noticed after 2 h of EVLP. Cellular and biomolecular changes in BAL fluid and histologic alterations were comparable among the three study groups. CONCLUSION: Preemptive treatment of donors with steroids but not macrolides improves gas exchange in a porcine lung injury model independently from its anti-inflammatory activity.

A model of ex vivo perfusion of porcine donor lungs injured by gastric aspiration: a step towards pretransplant reconditioning.

BACKGROUND: Evidence of aspiration remains a major reason for declining donor lungs contributing to current organ shortage. The aim of the present ex vivo lung perfusion (EVLP) study was to compare lungs injured by gastric juice (GJ) with normal lungs. METHODS: Pigs (32.3 ± 11.2 kg) were divided into two groups. Study group [GJ; n = 6] was intratracheally instilled with GJ, while sham bronchoscopy was performed in control group [C; n = 6]. Graft function was assessed during EVLP for 2 h. Oxygenation, aerodynamic, and hemodynamic parameters were recorded every 30 min. Wet to dry weight ratio (W/D) was calculated. Bronchoalveolar lavage was performed. Tissue samples were collected. RESULTS: Pulmonary vascular resistance was higher and pulmonary flow was lower in [GJ] versus [C] at T120; (P < 0.05). Mean airway pressure was higher in (P < 0.05) and compliance was lower (P < 0.001) in [GJ]. No differences in oxygenation were seen between groups. W/D of left lung in [GJ] after EVLP was slightly (P < 0.05) higher compared with the nonperfused right lung. More neutrophils were present in [GJ] before (P < 0.01) and after EVLP (P < 0.05). Histologic alterations were more prominent in [GJ], but did not worsen after EVLP. CONCLUSION: EVLP of injured lungs is possible for 2 h despite increased edema. This model could help to investigate ways to repair caustic lung injury during EVLP.

Medium-term outcome after lung transplantation is comparable between brain-dead and cardiac-dead donors.

BACKGROUND: Donation after cardiac death (DCD) to overcome the donor organ shortage is now moving into the clinical setting, but the medium-term outcome after DCD lung transplantation (LTx) remains largely unknown. METHODS: In this retrospective study, DCD LTx recipients (n = 21) were compared with a cohort of donation-after-brain-death (DBD) LTx recipients (n = 154) transplanted between February 2007 and July 2010. Immediate (post)operative outcome was evaluated by assessing need for peri-operative extracorporeal membrane oxygenation (ECMO), time to extubation, hospital discharge and primary graft dysfunction (PGD) within the first 48 hours. Survival, incidence of bronchiolitis obliterans syndrome (BOS), acute rejection (AR) and inflammatory markers in blood and in bronchoalveolar lavage (BAL) were assessed and compared over a median follow-up of 327 days for DCD and 531 days for DBD, showing no statistically significant difference (NS). RESULTS: There were no differences between groups with regard to patient characteristics except for a higher number of patients transplanted for obliterative bronchiolitis in the DCD group (4 of 21 vs 7 of 154; p < 0.05). In the DCD group, 2 of 21 patients died, vs 23 of 154 patients in the DBD group (NS). Actuarial survival rates at 6 months, 1 year and 3 years are 95%, 95% and 71% for the DCD group and 96%, 91% and 75% for the DBD group (NS). Three patients (14%) in the DCD group developed BOS vs 15 patients (10%) in the DBD group (NS). Survival and freedom from BOS were not different between the groups. AR, inflammatory markers and immediate (post)operative outcome also did not differ. CONCLUSIONS: In our experience, both early- and medium-term outcome in DCD lung recipients is comparable to that of DBD lung recipients. Use of lungs recovered from controlled donors after cardiac death is a safe option for expansion of the donor pool.

The impact of traffic air pollution on bronchiolitis obliterans syndrome and mortality after lung transplantation.

BACKGROUND: Approximately half of all patients who underwent a lung transplantation suffer from bronchiolitis obliterans syndrome (BOS), the clinical correlate of chronic rejection, within 5 years after transplantation. This prevalence is much higher than for other solid organ transplantations, possibly due to the lung's direct contact with the environment. The authors assessed the association between proximity of the home to major roads and BOS and mortality in a cohort of patients after lung transplantation. METHODS: The authors calculated hazard ratios for BOS and mortality in relation to proximity of the home to major roads, adjusting for relevant covariables, in 288 patients after lung transplantation at the Leuven University Hospital between 1997 and 2009 and with follow-up until August 2009. Inflammatory parameters in plasma and bronchoalveolar lavage were assessed in 207 patients. RESULTS: During follow-up, 117 (41%) patients developed BOS and 61 (21%) died. Patients who lived within 171 m of a major road (lowest tertile) were 2.06 (95% CI 1.39 to 3.05) times more likely to develop BOS and 2.20 (1.25 to 3.86) times more likely to die than patients living farther away. The adjusted hazard ratios of BOS and mortality were 0.57 and 0.72 for each 10-fold increase in distance from major roads. Proximity to a major road was inversely associated with plasma C-reactive protein levels, neutrophil percentage and interleukin-6 concentration in bronchoalveolar lavage. CONCLUSION: Traffic-related air pollution appears to constitute a serious risk of BOS and mortality after lung transplantation.

Donor cause of brain death and related time intervals: does it affect outcome after lung transplantation?

OBJECTIVE: It remains uncertain whether donor cause of brain death (DCBD) affects survival and freedom from bronchiolitis obliterans syndrome (BOS) after lung transplantation (LTx). In addition, it is unknown whether the length of time interval from brain insult to brain death [BI-BD] and from brain death to cold preservation [BD-CP] has an impact on outcome. METHODS: Medical charts of isolated lung transplant recipients from 400 consecutive donors were reviewed and classified according to DCBD: 190 vascular [V], 185 traumatic [T], 25 others [O]. Demographics were compared between donor groups. Hospital outcome, survival, and freedom from BOS in recipients were analyzed in relation to DCBD and related time intervals. RESULTS: Donor age, gender, and weight differed between donor groups (p<0.001, p<0.001, p<0.05; respectively). No differences in recipient hospital outcome, survival, and freedom from BOS were found between groups. [BD-CP] longer than 10h resulted in a survival advantage (69% vs 58% and 51% vs 42% at 5 and 10 years, respectively; p<0.05) and a reduced hazard risk (0.952) of dying after LTx; (p<0.05). Multivariable analysis failed to show a significant correlation between DCBD and [BI-BD] versus survival and BOS. CONCLUSION: DCBD and [BI-BD] do not affect survival and freedom from BOS after LTx. Lung recipients from donors certified brain dead with a time interval longer than 10h prior to organ preservation showed improved survival unrelated to BOS. This may result from longer and better donor management with reduced lung injury.

A porcine model to study ex vivo reconditioning of injured donor lungs.

BACKGROUND: Brain death rapidly results in lung injury making many cadaveric donors unsuitable for lung transplantation. The aim of this study was to develop a porcine model of lung injury as a first step to study mechanisms to ameliorate the pretransplant graft quality during ex vivo perfusion. MATERIALS AND METHODS: Male pigs (47 ± 8 kg) were divided into three groups: LPS-group [LPS] (n = 6) [instillation of lipopolysaccharides (15 mg/lung)]; saline-group [SAL] (n = 5) (50 mL saline/lung); and sham-group [SHAM] (n = 5). CT scans of the lungs were taken 17h before (T-17) and 31h after (T31) instillation. Broncho-alveolar lavage (BAL) was performed, and blood gases, hemodynamic, and aerodynamic parameters were measured at T 0 and T 50. Blood samples and temperature were taken at all time points. Pigs were sacrificed during cold pulmoplegia (T 50), and tissue samples were collected for histology. Wet lung weight was measured. RESULTS: Wet lung weight/body weight was higher in [LPS] versus [SAL] (P < 0.05). Total BAL cells were higher in [LPS] versus [SAL] and [SHAM] at T 50 (left: P < 0.001 and P < 0.01; right: P < 0.01 and P < 0.001). More neutrophils were present in BAL of [LPS] at T 50 versus T 0 (left: P < 0.001; right: P < 0.01). [LPS] demonstrated more ground glass opacities (GGO) on CT at T 31 compared with [SAL] and [SHAM] (P < 0.05). Histologically, more interstitial hemorrhage was observed in [LPS] versus [SAL] and [SHAM](P < 0.01). Neutrophils in blood increased and lymphocytes decreased in [LPS] versus [SAL] (P < 0.05). No differences were observed in hemodynamic and aerodynamic parameters and in saturation between groups at T 50. CONCLUSIONS: LPS instillation caused inflammation with more cells in BAL, changes on CT, and histology. However, no physiologic changes occurred.

A porcine model of acute lung injury by instillation of gastric fluid.

BACKGROUND: About 15% of donor lungs are declined because of aspiration contributing to current organ shortage. The aim was to develop a porcine lung injury model by gastric juice (GJ) instillation to study different pretransplant treatment strategies. MATERIALS AND METHODS: Pigs (n = 6/group) were anesthetized and monitored. At T0 bronchoalveolar lavage (BAL) was performed followed by instillation of 4 mL/kg GJ or saline solution (SAL). Hemodynamics, aerodynamics and oxygenation were recorded for two hours. Serum samples were collected. At T120 a second BAL was performed. CT scans of explanted, inflated lungs were taken, tissue samples were collected and wet/dry weight ratio (W/D) was calculated. Pepsin and bile acids were measured in BAL. IL-8, CRP and MMP-9 were measured in serum and in BAL. RESULT: Oxygenation and lung compliance was lower in [GJ] versus [SAL] (P < 0.01 and P < 0.001, respectively). More consolidation areas were noticed on CT in GJ versus SAL (P < 0.01). Hemorrhage, edema and neutrophil inflammation were seen on histology in [GJ] (P < 0.01, P < 0.001, P < 0.001, respectively). BAL neutrophils, pepsin, bile acids, and IL-8 (P < 0.05) increased in [GJ]. W/D was higher in [GJ] versus SAL (P < 0.001). CONCLUSION: Instillation of GJ in pig lungs caused acute lung injury with impaired oxygenation and increased inflammation in BAL, on histology, and on imaging. This model holds promise to assess the efficacy of a broad range of treatment strategies including ex vivo lung perfusion (EVLP).

Long-term azithromycin therapy for bronchiolitis obliterans syndrome: divide and conquer?

BACKGROUND: Azithromycin may reverse or halt the decline of pulmonary function (FEV(1)) in bronchiolitis obliterans syndrome (BOS). In this study we investigated the effects of long-term azithromycin treatment in lung transplant recipients with BOS. METHODS: A retrospective, observational, cohort study was performed on 107 patients with BOS (Stages 0p/1/2/3, n = 23/62/20/2), who were treated with azithromycin for 3.1 ± 1.9 years. Patients were evaluated 6.3 ± 3.8 years after transplantation and assessed for evolution of FEV(1), bronchoalveolar lavage neutrophilia and overall survival after initiation of azithromycin. Survival curves were analyzed using the log-rank test. Cox proportional hazard survival regression analysis was performed to estimate hazard ratios of clinical variables predicting outcome. RESULTS: FEV(1) increased ≥ 10% after 3 to 6 months of treatment in 40% of patients, of whom 33% later redeveloped BOS. FEV(1) further declined in 78% and stabilized in 22% of the remaining non-responders. Pre-treatment neutrophilia was higher in responders: 29.3% (9.3% to 69.7%) vs 11.5% (2.9% to 43.8%) (p = 0.025), in whom it significantly decreased to 4.2% (1.8% to 17.6%) (p = 0.041) after 3 to 6 months of azithromycin. Responders demonstrated better survival compared with non-responders (p = 0.050), with 6 and 21 patients, respectively, dying during follow-up (p = 0.027). Multivariate analysis identified initial azithromycin response and earlier post-transplant initiation of azithromycin to be protective for both BOS progression/relapse (hazard ratio [HR] = 0.12 [95% confidence interval 0.05 to 0.28], p < 0.0001; and HR = 0.98 [95% confidence interval 0.97 to 0.98], p < 0.0001, respectively) and retransplantation/death during follow-up (HR 0.10 [95% confidence interval 0.02 to 0.48], p = 0.004; and HR 0.96 [95% confidence interval 0.95 to 0.98], p < 0.0001, respectively). CONCLUSIONS: Long-term azithromycin benefits pulmonary function and survival in BOS, particularly in patients with increased lavage neutrophilia.

Early outcome after lung transplantation from non-heart-beating donors is comparable to heart-beating donors.

BACKGROUND: The use of non-heart-beating donors (NHBD) to overcome organ shortage is moving into the clinic. In 2007, 5 of 51 lung transplantations (LTx) in our center were performed with lungs from controlled NHBD. METHODS: Our aim was to describe these 5 NHBD LTx recipients and compare early outcome (