Ex Vivo Perfusion With Methylprednisolone Attenuates Brain Death-induced Lung Injury in Rats

Abstract

The onset of brain death (BD) leads to the deterioration of potential donor lungs. Methylprednisolone is considered to increase lung oxygenation capacity and enhance the procurement yield of donor lungs, when applied in situ, during donor management. However, whether BD-induced lung damage is ameliorated upon treatment with methylprednisolone during acellular ex vivo lung perfusion (EVLP), remains unknown. We aimed to investigate whether the quality of lungs from brain-dead donors improves upon methylprednisolone treatment during EVLP.

Methods: Rat lungs were randomly assigned to 1 of 3 experimental groups (n = 8/group): (1) healthy, directly procured lungs subjected to EVLP; (2) lungs from brain-dead rats subjected to cold storage and EVLP; and (3) lungs from brain-dead rats subjected to cold storage and EVLP with 40 mg methylprednisolone added to the perfusate. Ventilation and perfusion parameters, histology, edema formation, metabolic profile, and inflammatory status of lungs were investigated.

Results: Methylprednisolone treated lungs from brain-dead donors improved positive inspiratory pressures needed to maintain tidal volumes of 7 mL/kg of body weight, which was 25.6 ± 5.8 cm H₂O in untreated lungs and 18.0 ± 3.0 cm H₂O in methylprednisolone treated lungs, after 6 h EVLP. Furthermore, dynamic lung compliance increased upon methylprednisolone treatment, with values of 0.11 ± 0.05 mL/cm H₂O versus 0.18 ± 0.04 mL/cm H₂O after 6 h of EVLP. Methylprednisolone treatment ameliorated the amount of lung edema, as corroborated by a reduction of 0.7 in the wet/dry ratio. Although glucose consumption levels were comparable, the BD-induced cumulative lactate production decreased from 0.44 ± 0.26 to 0.11 ± 0.16 mmol/L upon methylprednisolone treatment. Finally, BD-induced inflammatory status was reduced upon methylprednisolone treatment compared to untreated lungs from brain-dead donors, as reflected by lower proinflammatory gene expression levels of IL-1β, IL-6 and MCP-1, and IL-6 perfusate levels.

Conclusions: We showed that methylprednisolone treatment during EVLP attenuates BD-induced lung injury.

FIGURE 1.

Outline of the study. Lungs from donor rats were randomly assigned to 1 of 3 experimental groups: (1) healthy, directly procured lungs subjected to 6 h EVLP; (2) lungs from brain-dead rats (BD sustained for 3 h) subjected to 1 h CS and 6 h EVLP; and (3) lungs from brain-dead rats (BD sustained for 3 h) subjected to 1 h CS and 6 h EVLP with 40 mg methylprednisolone added to the perfusate. BD, brain death; CS, cold storage; EVLP, ex vivo lung perfusion.

FIGURE 2.

Ventilation and perfusion parameters during EVLP. Lungs from donor rats were randomly assigned to 1 of 3 experimental groups: (1) healthy, directly procured lungs subjected to 6 h EVLP; (2) lungs from brain-dead rats (BD sustained for 3 h) subjected to 1 h CS and 6 h EVLP; and (3) lungs from brain-dead rats (BD sustained for 3 h) subjected to 1 h CS and 6 h EVLP with 40 mg methylprednisolone (Pred) added to the perfusate. A, Positive inspiratory pressures required to maintain tidal volumes of 7 mL/kg of body weight. B, Dynamic lung compliance of lungs during EVLP. C, Oxygenation status of lungs during EVLP, reflected by Pao₂/FiO₂ ratio. D, Perfusion flow of lungs during EVLP. *P < 0.05 in BD lungs vs BD + Pred lungs, **P < 0.01 in BD lungs vs BD + Pred lungs, ^#P < 0.05 in BD lungs vs healthy lungs, ^##P < 0.01 in BD lungs vs healthy lungs. BD, brain death; CS, cold storage; EVLP, ex vivo lung perfusion.

FIGURE 3.

Lung morphology of lungs after EVLP. Lungs from donor rats were randomly assigned to 1 of 3 experimental groups: (1) healthy, directly procured lungs subjected to 6 h EVLP; (2) lungs from brain-dead rats (BD sustained for 3 h) subjected to 1 h CS and 6 h EVLP; and (3) lungs from brain-dead rats (BD sustained for 3 h) subjected to 1 h CS and 6 h EVLP with 40 mg methylprednisolone (Pred) added to the perfusate. Lung morphology scores were assessed after 6 h of EVLP by means of hematoxylin and eosin staining. A, Quantification of lung morphology scores in hematoxylin and eosin stained lung slides. B–D, Representative hematoxylin and eosin stained slices of healthy donor lungs, untreated lungs from brain-dead donors, and methylprednisolone treated lungs from brain-dead donors, after 6 h of EVLP. ^#P < 0.05 in BD lungs vs healthy lungs. BD, brain death; CS, cold storage; EVLP, ex vivo lung perfusion.

FIGURE 4.

Metabolic profile of lungs during EVLP. Lungs from donor rats were randomly assigned to 1 of 3 experimental groups: (1) healthy, directly procured lungs subjected to 6 h EVLP; (2) lungs from brain-dead rats (BD sustained for 3 h) subjected to 1 h CS and 6 h EVLP; and (3) lungs from brain-dead rats (BD sustained for 3 h) subjected to 1 h CS and 6 h EVLP with 40 mg methylprednisolone (Pred) added to the perfusate. A, Cumulative glucose consumption by lungs during EVLP. B, Cumulative lactate production by lungs during EVLP. *P < 0.05 in BD lungs vs BD + Pred lungs, ^#P < 0.05 in BD lungs vs healthy lungs. BD, brain death; CS, cold storage; EVLP, ex vivo lung perfusion.

FIGURE 5.

Inflammatory status of lungs during EVLP. Lungs from donor rats were randomly assigned to 1 of 3 experimental groups: (1) healthy, directly procured lungs subjected to 6 h EVLP; (2) lungs from brain-dead rats (BD sustained for 3 h) subjected to 1 h CS and 6 h EVLP; and (3) lungs from brain-dead rats (BD sustained for 3 h) subjected to 1 h CS and 6 h EVLP with 40 mg methylprednisolone (Pred) added to the perfusate. A–E, mRNA gene expression levels of proinflammatory mediators (A) TNF-α, (B) IL-1β, (C) IL-6, (D) MCP-1, and (E) C3 in lung tissue measured after 6 h of EVLP. F, Concentration of IL-6 protein in perfusate over time. *P < 0.05 in BD lungs vs BD + Pred lungs, **P < 0.01 in BD lungs vs BD + Pred lungs, ^{# #}P < 0.01 in BD lungs vs healthy lungs, ^xP < 0.05 in healthy vs BD + Pred lungs, ^xxP < 0.01 in healthy vs BD + Pred lungs. BD, brain death; CS, cold storage; EVLP, ex vivo lung perfusion.