Pyrrolidine dithiocarbamate administered during ex-vivo lung perfusion promotes rehabilitation of injured donor rat lungs obtained after prolonged warm ischemia

Abstract

Damaged lung grafts obtained after circulatory death (DCD lungs) and warm ischemia may be at high risk of reperfusion injury after transplantation. Such lungs could be pharmacologically reconditioned using ex-vivo lung perfusion (EVLP). Since acute inflammation related to the activation of nuclear factor kappaB (NF-κB) is instrumental in lung reperfusion injury, we hypothesized that DCD lungs might be treated during EVLP by pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB. Rat lungs exposed to 1h warm ischemia and 2 h cold ischemia were subjected to EVLP during 4h, in absence (CTRL group, N = 6) or in presence of PDTC (2.5g/L, PDTC group, N = 6). Static pulmonary compliance (SPC), peak airway pressure (PAWP), pulmonary vascular resistance (PVR), and oxygenation capacity were determined during EVLP. After EVLP, we measured the weight gain of the heart-lung block (edema), and the concentration of LDH (cell damage), proteins (permeability edema) and of the cytokines IL-6, TNF-α and CINC-1 in bronchoalveolar lavage (BAL), and we evaluated NF-κB activation by the degree of phosphorylation and degradation of its inhibitor IκBα in lung tissue. In CTRL, we found significant NF-κB activation, lung edema, and a massive release of LDH, proteins and cytokines. SPC significantly decreased, PAWP and PVR increased, while oxygenation tended to decrease. Treatment with PDTC during EVLP inhibited NF-κB activation, did not influence LDH release, but markedly reduced lung edema and protein concentration in BAL, suppressed TNFα and IL-6 release, and abrogated the changes in SPC, PAWP and PVR, with unchanged oxygenation. In conclusion, suppression of innate immune activation during EVLP using the NF-κB inhibitor PDTC promotes significant improvement of damaged rat DCD lungs. Future studies will determine if such rehabilitated lungs are suitable for in vivo transplantation.

Fig 1. Experimental protocol.

Rats were sacrificed by exsanguination and perfusion cannulae were inserted into the pulmonary artery and the left atrium. The chest was left open for one hour at room temperature (warm ischemic time), with the lungs deflated. The lungs were then flushed with cold Perfadex® solution, inflated (5ml·kg^-1, FiO₂ 0.5), and kept 2 hours in 4°C Perfadex® (cold ischemic time). The heart-lung blocks were then weighted (lung weight 1) mounted into a rat EVLP system, and perfused for a total of 4 hours, either with Steen® solution alone (Control group, CTRL, N = 6) or with Steen® solution supplemented with pyrrolidine dithiocarbamate ammonium (PDTC, C₅H₉NS₂-NH₃), at a concentration of 2.5g·L^-1 (PDTC group, N = 6). At the end of EVLP protocol, the heart-lung blocks were once again weighted (lung weight 2), a bronchoalveolar lavage was performed through the tracheal cannula, and the left lung was frozen for further analysis.

Fig 2. LDH activity in BAL fluid and protein carbonyls in lung tissue homogenates.

A. LDH activity in BAL fluid. LDH activity was measured as an index of cellular necrosis, expressed in arbitrary units (A.U.). B: protein carbonyls were measured as an index of oxidative modifications in lung tissue, normalized to the concentration of lung proteins. BASE: BAL fluid and lung tissue were obtained from normal rats sacrificed without any intervention (Normal, baseline values, N = 3); CTRL: Ischemia followed by EVLP with Steen® solution alone (N = 6); PDTC: Ischemia followed by EVLP with PDTC treatment (N = 6). Means ± s.e.m.* p<0.05 (ANOVA followed by Tukey’s test).

Fig 3. Concentrations of inflammatory cytokines in the BAL fluid.

BAL fluid concentration of IL-6 (A), TNFα (B) and CINC-1 (C), expressed in ng^.ml^-1 BAL fluid. BASE group: BAL fluid was obtained from normal rats sacrificed without any intervention (normal values, N = 3); CTRL: Ischemia followed by EVLP with Steen® solution alone (N = 6); PDTC: Ischemia followed by EVLP with PDTC treatment (N = 6). Means ± s.e.m.* p<0.05. (ANOVA followed by Tukey’s test).

Fig 4. Indices of lung edema.

A. Weight gain of the heart-lung blocks expressed in grams, represents the difference between weight of the heart-lung blocks measured before and at the end of EVLP. B. The concentration of proteins in the BAL fluid (in mg^.ml^-1 BAL fluid) was measured as an index of high permeability edema. BASE: BAL fluid and was obtained from normal rats sacrificed without any intervention (Normal, baseline values, N = 3); CTRL: Ischemia followed by EVLP with Steen® solution alone (N = 6); PDTC: Ischemia followed by EVLP with PDTC treatment (N = 6). Means ± s.e.m.* p<0.05 (Heart-lung block weight gain: unpaired t test; Proteins in BAL fluid: ANOVA followed by Tukey’s test).

Fig 5. Static pulmonary compliance and peak airway pressure during EVLP.

Static pulmonary compliance (A) and peak airway pressure (B) were computed 60 minutes after EVLP initiation (baseline values), and every 30 minutes thereafter. CTRL: Ischemia followed by EVLP with Steen® solution alone (N = 6); PDTC: Ischemia followed by EVLP with PDTC treatment (N = 6). Means ± s.e.m. † p < 0.05 vs baseline value; * p<0.05 PDTC vs CTRL (two way ANOVA with Bonferroni’s adjustments for multiples comparisons)

Fig 6. Pulmonary vascular resistance and oxygenation index during EVLP.

Pulmonary vascular resistance (A) was calculated as (mean pulmonary artery pressure - left atrial pressure)/perfusion flow). The oxygenation capacity (B) was calculated as the difference in the partial pressure of O₂ between the effluent and the affluent arms of the EVLP circuits. The vascular resistance and oxygenation capacity were computed after 60 minutes of EVLP (baseline values), and every 30 minutes thereafter. Means ± s.e.m. † p < 0.05 vs baseline value; * p<0.05 PDTC vs CTRL (two way ANOVA with Bonferroni’s adjustments for multiples comparisons).

Fig 7. Phosphorylation and degradation of IκBα in lung tissue.

Western immunoblots of IκBα and phospho-IκBα in all lungs (A). The graph depicts the ratio of the densitometric analysis of phospho-IκBα and IκBα (B). Means ± s.e.m; ** p<0.01 (ANOVA followed by Tukey’s test).