Cromolyn ameliorates acute and chronic injury in a rat lung transplant model

Abstract

Background: Mast cells have been associated with obliterative bronchiolitis (OB) in human pulmonary allografts, although their role in the development of OB remains unknown.

Methods: In this study, we evaluated the role of mast cells in pulmonary allograft rejection using an orthotopic rat pulmonary allograft model that utilizes chronic aspiration of gastric fluid to reliably obtain OB. Pulmonary allograft recipients (n = 35) received chronic aspiration of gastric fluid with (n = 10) and without (n = 16) treatment with a mast cell membrane stabilizer, cromolyn sodium, or chronic aspiration with normal saline (n = 9) as a control.

Results: The acute graft injury associated with long ischemic time in the model (6 hours total ischemic time; typical acute graft injury rate ~30%) was apparently blocked by cromolyn, because peri-operative mortality associated with the acute graft injury was not observed in any of the animals receiving cromolyn (p = 0.045). Further, the rats receiving cromolyn developed significantly fewer OB lesions than those treated with gastric fluid alone (p < 0.001), with a mean reduction of 46% of the airways affected.

Conclusions: These findings provide impetus for further studies aimed at elucidating the effects of cromolyn and the role of mast cells in pulmonary allotransplantation.

Keywords: aspiration; gastric fluid; mast cells; obliterative bronchiolitis; pulmonary allograft.

Figure 1

Peri-operative morbidity and mortality of recipients treated with cromolyn sodium. In this experiment, 11 of 36 rats receiving lungs subjected to prolonged ischemia developed peri-operative morbidity and mortality with histologic evidence of parenchymal necrosis and diffuse alveolar hemorrhage. In contrast, no peri-operative morbidity or mortality developed in those recipients treated with pre-operative cromolyn, despite receiving lungs subjected to prolonged ischemia. Analysis using a 2 × 2 contingency table showed a significant improvement in peri-operative complications and death after treatment with cromolyn (p = 0.045).

Figure 2

Gross morphology and lung compliance of pulmonary allografts 9 to 10 weeks after transplantation. After 8 weeks of gastric fluid (GF) aspiration, most of the transplanted lungs (arrow) had discoloration, consolidation and reduced size (A). On the other hand, the majority of allografts from recipients treated with cromolyn sodium expanded to >70% of normal lung volume (see Methods) and had normal lung contours (B), despite 8 weeks of GF aspiration. The allografts in the control group, receiving aspiration with normal saline, were grossly normal (C). The percentage of gross consolidation in the GF aspiration group was significantly higher than that in the GF aspiration plus cromolyn group (p = 0.003; analysis with a 2 × 2 contingency table) and the control group (p < 0.001; analysis with a 2 × 2 contingency table). There was no significant difference between the GF aspiration plus cromolyn group and the control group (p = 0.263; analysis with a 2 × 2 contingency table) (D). Pulmonary allografts in the GF aspiration plus cromolyn and control groups had similar compliance, whereas worse compliance was noted in allografts in the GF aspiration group (E). The volume of allografts treated with GF aspiration plus cromolyn at a pressure of 380 mm H₂O was, on average, increased by >100% compared with grafts receiving aspiration without cromolyn (mean ± SE: 1.239 ± 0.361 ml vs 0.448 ± 0.255 ml; p = 0.049, 1-tailed t-test).

Figure 3

Histologic analysis of the small airways in the pulmonary allografts. An example of the OB lesions that developed in allografts exposed to 8 weekly aspirations of gastric fluid (GF) is shown (the arrows indicate the smooth muscle of this bronchiole). The lesions were characterized by epithelial damage and sub-epithelial fibroproliferation (A). In contrast, most bronchioles in lungs from the GF aspiration plus cromolyn and control (B, C) groups lacked characteristic features of OB. Quantitative analysis revealed that the fraction of airways affected was significantly higher in the GF aspiration group than in the GF aspiration plus cromolyn (p < 0.001) and the control (p < 0.001) groups. There was no significant difference between the percentage of airways affected in the allografts of the GF aspiration plus cromolyn group and the control group (p = 0.258) (D). Bar = 50 μm; hematoxylin and eosin stain.

Figure 4

Histologic analysis of pulmonary parenchyma in the pulmonary allografts. Pulmonary allografts exposed to 8 weekly aspirations of gastric fluid (GF) developed severe fibrosis and destruction of the parenchyma (A, D). Recipients given GF aspiration plus cromolyn developed mild to moderate fibrosis of pulmonary parenchyma in the allografts (B, E). Expansion of the parenchyma and normal architecture of the allografts were noted in the control group, which was exposed to 8 weekly aspirations of normal saline (C, F). Semi-quantitative analysis of parenchymal fibrosis revealed that allografts treated with GF aspiration plus cromolyn had significantly less parenchymal fibrosis than allografts treated with GF aspiration without cromolyn (p = 0.031) (G). Bar = 200 μm; (A–C) hematoxylin and eosin stain; (D–F) Masson trichrome stain.

Figure 5

Mast cells in the small bronchioles. Serial sections using hematoxylin and eosin stain (A) and toluidine blue stain (B) show an OB lesion in a pulmonary allograft exposed to 8 weekly aspirations of gastric fluid (GF). In (A), arrows denote the smooth muscle of the bronchiole, and in (B) arrows indicate mast cells associated with the bronchiole. Different stages of degranulation of mast cells were noted in the bronchiole using toluidine blue staining (C–E). The number of mast cells per small bronchiole increased significantly in animals aspirated with GF, regardless of cromolyn treatment (GF aspiration vs control: p = 0.005; GF aspiration plus cromolyn vs control: p = 0.024). There was no significant difference between the number of mast cells per bronchiole in animals with and without treatment with cromolyn (GF aspiration vs GF aspiration plus cromolyn: p = 0.176) (F). A positive correlation was observed between OB and the number of mast cells in allografts from the GF aspiration group (r = 0.709, p = 0.002), but not in allografts from the GF aspiration plus cromolyn group (r = −0.225, p = 0.532) or the control group (r = −0.297, p = 0.439) (G). Bar = 200 μm in (A) and (B) and 5 μm in (C)–(E).