Enalapril mitigates focal alveolar lesions, a histological marker of late pulmonary injury by radiation to the lung

Abstract

The goal of our study was to identify a histological marker for testing countermeasures for mitigation of late radiation injury to the lung. Pulmonary fibrosis is currently the best described "late effect" in survivors of acute radiation pneumonitis. However, robust fibrosis does not develop in some rodent strains for years after a single dose of radiation to the whole thorax. We observed radiation-associated focal alveolar lesions that were rich in giant cells and macrophages containing cholesterol clefts in the lungs of irradiated WAG/RijCmcr rats. These lesions were first observed after pneumonitis, around 21 weeks after receiving a radiation dose of 13 Gy to the thorax but not until 71 weeks in unirradiated rats. The number of cholesterol clefts increased with time after irradiation through 64 weeks of observation, and at 30 weeks after 13 Gy, cholesterol clefts were associated with several indices of deterioration in lung function. The number of cholesterol clefts in irradiated lung sections were reduced by the angiotensin converting enzyme (ACE) inhibitor enalapril (25-42 mg/m²/day) from 18.7 ± 4.2/lung section to 6.8 ± 2.4 (P = 0.029), 5.2 ± 1.9 (P = 0.0051) and 6.7 ± 1.9 (P = 0.029) when the drug was started at 1 week, 5 or 15 weeks after irradiation, respectively, and continued. Similar lesions have been previously observed in the lungs of one strain of irradiated mice and in patients following radiotherapy. We propose that alveolar lesions with cholesterol clefts may be used as a histological marker of the severity of radiation lung injury and to study its mitigation in WAG/RijCmcr rats.

FIG. 1

Histology. Representative images from typical alveolar lesions (left) and lung parenchyma (right) are shown after (panel A) H&E staining with arrows pointing to empty cholesterol clefts. Panel B: H&E stained section of lung parenchyma with typical epithelial cell metaplasia (see arrows). Panel C: Trichrome stained section with a circle enclosing an alveolar lesion that was analyzed for morphometric comparisons of the ratio of red (noncollagenous):blue (collagen) staining. Panel D: Graphical representation of red:blue ratio in alveolar lesions (dotted bar) and lung parenchyma (open bar). Values are presented as mean ± SEM. n = number of rats per group. There was no statistically significant difference in the red:blue stain ratio between these 2 groups. Panel E: Whole-mount lung section immunostained with anti-CD 68 antibody under low magnification. Arrows point to islands of positively stained macrophages. Bars represent 20 μm in panel A, 100 μm in panel B, 50 μm in panel C and 5 mm in panel E.

FIG. 2

Immunohistochemistry. Representative images from typical alveolar lesions (left) and lung parenchyma (right) immunostained with antibodies from irradiated rats as follows, panel A: CD 68 (macrophages), panel B: myeloperoxidase (macrophages and neutrophils), panel C: CD 43 (T cells), panel D: CD 79a (B cells), panel E: tryptase (mast cells), panel F: CD 31 (vascular endothelial cells), panel G: keratin-7 (epithelial cells) and panel H: smooth muscle alpha actin (smooth muscle cells). Bars represent 20 μm. Note macrophages stained positive for CD-68 (panel A) and myeloperoxidase (panel B) appear more abundant in alveolar lesions compared to the parenchyma, highlighting areas that are clearly visible in the whole-mount section in Fig. 1E. Arrows in panels F and G point to a narrowed blood vessels and airway, respectively.

FIG. 3

Scatter plots of cholesterol cleft counts/whole-mount lung sections. Open circles represent unirradiated (control) rats corresponding to 30 (n = 8), 43 (n = 6), 57 (n = 3) and 71 (n = 6) weeks after irradiation in the irradiated groups. No cholesterol clefts were observed before 71 weeks in these rats. Closed circles represent clefts at 21 (n = 8), 25 (n = 8), 30 (n = 8), 43 (n = 7) and 64 (n = 5) weeks after a radiation dose of 13 Gy. All points positioned exactly on the vertical axis, except for some zero values shown below zero for clarity. The solid black (13 Gy) and dashed gray (0 Gy) lines connect mean values in each group.

FIG. 4

Scatter plots of cholesterol cleft counts/whole-mount lung sections. Open triangles represent results from lungs of rats (n = 8) after 56 weeks after irradiation with 5 Gy to the thorax. Solid black triangles represent results from lungs (n = 7) after 53 weeks after irradiation with 10 Gy to the thorax. Open stars represent results from lungs after 33 weeks (n = 6) and 41 weeks (n = 10) after irradiation with 12 Gy to the thorax. No cholesterol clefts were observed after a radiation dose of 5 Gy at 56 weeks or 12 Gy at 33 weeks. All points positioned exactly on the vertical axis, except for some zero values shown below zero for clarity. The line in each group represents the mean value.

FIG. 5

Graph showing mean ± SEM body weights of rats in grams at 30 weeks after irradiation. Open bar represents unirradiated (control) rats (n = 8). Solid black bar represents rats with radiation doses of 13 Gy to the thorax (n = 8). Gray bars represent thorax-irradiated rats (13 Gy) given enalapril started at different times after irradiation, as labeled in the figure. Solid gray bars (n = 8) depict the weight of rats treated with 60 mg/L enalapril and hatched bars (n = 8) show the weight of rats treated with 30 mg/L enalapril. Unirradiated rats had higher body weight compared to all other groups (*P < 0.05, ANOVA with Holm-Sidak method). No difference was observed for results between the other groups.

FIG. 6

Scatter plots of cholesterol cleft counts at 30 weeks in unirradiated rats (open circles) and after 13 Gy irradiation to the whole thorax (gray circles). The black circles represent 13 Gy + enalapril 60 mg/L started at 1, 5, 10, 15, and 20 weeks after irradiation and continued to 30 weeks (210 days). n = 8 rats/group. The line in each group represents the mean value.