In the face of chronic aspiration, prolonged ischemic time exacerbates obliterative bronchiolitis in rat pulmonary allografts

Abstract

Aspiration of gastric fluid into the lung mediates the development of obliterative bronchiolitis (OB) in orthotopic WKY-to-F344 rat pulmonary transplants that have been subjected to immunosuppression with cyclosporine. However, the contribution of ischemic time to this process remains unknown. In this study, the effect of long (n = 16) and short (n = 12) ischemic times (average of 6 h and of 73 min, respectively) on rat lung transplants receiving aspiration of gastric fluid was assessed. Long ischemic times (LIT) led to significantly (p < 0.05) greater development of OB (ratio of OB lesions/total airways = 0.45 ± 0.07, mean ± standard error) compared to short ischemic times (ratio = 0.19 ± 0.05). However, the development of OB was dependent on aspiration, as controls receiving aspiration with normal saline showed little development of OB, regardless of ischemic time (p < 0.05). These data suggest that LIT, while insufficient by itself to lead to OB, works synergistically with aspiration of gastric fluid to exacerbate the development of OB.

Figure 1. Time course of the experiment

Transplanted rats received aspiration of 0.5 mL/kg gastric fluid or normal saline once weekly into the left (transplanted) lung for 8 weeks starting one week posttransplantation. The rats were sacrificed one week after the last aspiration.

Figure 2. Gross morphology of lung allografts 9 weeks after transplantation

Lungs were exposed to either long (A and C) or short (B and D) ischemic time, and to aspiration with either gastric fluid (A and B) or normal saline (C and D). In the recipients receiving chronic exposure of gastric fluid, most of the allografts (arrow) developed shrinkage, discoloration and consolidation (A, B). In the animals with normal saline treatment, the majority of the left lung grafts were grossly normal (C, D).

Figure 3. Obliterative bronchiolitis developed in the allografts with treatment of gastric fluid

Lungs were exposed to either long (LIT; panels A and C) or short (SIT; panels B and D) ischemic time, and to aspiration with either gastric fluid (GF; panels A and B) or normal saline (NS; panels C and D). Sections were procured and stained with hematoxylin and eosin as described in the “Materials and Methods.” (A) Submucosal fibroproliferation, epithelial damage and regional mononuclear cell infiltration led to near-complete obliteration of bronchioles in an allograft of Allo LIT GF animals. (B) Fibrosis and inflammatory cell infiltration of the submucosa caused narrowing of small airways in one of the Allo SIT GF rats. (C, D) The epithelium and lumen remained normal in the Allo LIT and Allo SIT animals with treatment of normal saline (E) Quantitative analysis revealed significantly more OB lesions in Allo LIT GF grafts. The bar represents 50 μm.

Figure 4. Histological fibrosis in the parenchyma of the allografts with chronic exposure of gastric fluid

Lungs were exposed to either long (LIT; panels A and C) or short (SIT; panels B and D) ischemic time, and to aspiration with either gastric fluid (GF; panels A and B) or normal saline (NS; panels C and D). Sections were procured and stained with hematoxylin and eosin as described in the “Materials and Methods.” Infiltration of inflammatory cells and severe fibrosis with narrowing of the airways developed in the (A) Allo LIT GF and (B) Allo STI GF grafts. The remnants of gastric fluid can be seen (B, arrow). Expansion of the alveoli and patent airways were noted in the majority of the LIT and SIT allografts with normal saline treatment (C, D). (E) Lung parenchyma in the allografts with chronic exposure of gastric fluid revealed significantly more fibrosis than that in the allografts with treatment of normal saline. The bar represents 100 μm.

Figure 5. The effect of chronic gastric fluid exposure to the isograft

Lungs were exposed to either long (LIT) or short (SIT) ischemic time and to aspiration with gastric fluid (GF). Representative LIT grafts are shown in panels A–C. Consolidation and discoloration were noted in the majority of isografts receiving gastric fluid aspiration (A). Severe fibrosis of the parenchyma was observed, but patent bronchioles with intact epithelium were still seen (B, hematoxylin and eosin stain; C, Masson trichrome stain). Severe parenchyma fibrosis with chronic exposure to gastric fluid developed in most isografts (D). Relatively few OB-like lesions were seen in isografts (E) and no noticeable difference in parenchyma fibrosis or in the prevalence of OB lesions was noted in between the groups with long versus short ischemic time (D and E). The bar represents 100 μm.