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Pathology of Diacetyl and 2,3-Pentanedione Airway Lesions in A Rat Model of Obliterative Bronchiolitis

Gordon P. Flake, and Daniel L.Morgan.
Toxicology (2017) DOI: https://doi.org/10.1016/j.tox.2016.10.013 PMID: 27984136


Publication


Abstract

Inhalation of diacetyl vapors by workers has been associated with obliterative bronchiolitis (OB), a poorly understood fibroproliferative disease of the small airways. Significant insights into the pathogenesis of OB have been obtained through the use of a rat model. Inhalation exposure of rats to diacetyl or 2,3-pentanedione, a related flavoring agent, can cause severe injury to the airway epithelium and underlying basement membrane. Repeated exposure to diacetyl or 2,3-pentanedione leads to aberrant repair, fibroproliferation and partial to complete occlusion of the airway lumen. Fibroproliferative lesions in rat airways were found to include both intraluminal polyps and circumferential intramural lesions. Intraluminal polyps have been observed to form secondary attachments spanning the airway lumen causing increasing obstruction. These airway lesions in rats are accompanied by inflammation in the form of peribronchial and perivascular infiltrates of lymphocytes, eosinophils and neutrophils. Diacetyl-induced OB lesions in the rat are similar to OB lesions in humans and provide a good model for studying the pathogenesis of this disease.

Figures


Figure 1. Epithelial Injury and Response.

Male Wistar Han rats were exposed to 200 ppm 2,3-pentanedione 6 h/day, 5 days/week for 2 weeks. A) Ulceration of the bronchial mucosa, with a linear zone of fibrinoid necrosis (long arrow), and a few macrophages and neutrophils on the surface (short arrow). H&E. B) Regenerating bronchial epithelium in the form of elongate, flattened cells on the right (arrow) and cuboidal cells on the left (arrowhead). H&E. C) Respiratory epithelial hyperplasia, characterized by increased height and cellularity (compare to epithelium of control in inset). 200 ppm 2,3-pentanedione followed by 2-week recovery. H&E. D) Squamous metaplasia, with atypia. The multilayered epithelium contains cells with nuclear enlargement and hyperchromasia, and flattening at the surface. H&E. E) More extensive epithelial ulceration, tissue disruption, neutrophilic exudate on surface, and macrophages in tissue. In this type of lesion, it is likely that the basement membrane of the mucosa has been disrupted, which might lead to subsequent fibrosis. H&E. F) Immunohistochemical stain for laminin to highlight the basement membrane of the mucosa (arrows). Note the discontinuity of the basement membrane between the arrowheads, associated with proliferation of fibrous tissue that has protruded through the defect to form a fibrous polyp (asterisk) in the bronchial lumen. Original objective magnification: A, 10×; B, 20×; C, 20×; D, 20×; E, 10×; F, 10×. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Figure 2. Intraluminal (Polypoid) Fibrosis.

Male Wistar Han rats were exposed to 2,3-pentanedione 6 h/day, 5 days/week for 2 weeks. A) Early intraluminal fibrosis, largely filling the lumen of a small bronchus. Note the myxoid stroma of both the intraluminal (arrowhead) and the intramural (arrow) fibrosis, indicative of early lesions. 150 ppm 2,3-pentanedione, H&E. B) Developing, intraluminal polypoid fibrosis, still largely myxoid (arrows). 200 ppm 2,3-pentanedione, H&E. C) Masson trichrome stain of lesion in B shows very little blue staining within the polypoid tissue, indicating the absence of mature collagen. D) Alcian blue/PAS stain of lesion in B displays extensive blue-green staining of the fibrosis, indicative of abundant stromal mucopolysaccharides. E) Very large area of intraluminal fibrosis, consisting predominantly of mature collagen in the stroma (asterisk). Note multiple areas of secondary attachment of the polyp to the epithelium of the bronchial wall (arrows). 175 ppm 2,3-pentanedione, H&E. F) Very large sessile, polypoid fibrous growth which extends both proximally and distally (arrowheads), and largely occludes the lumen of the bronchial branch on the left (arrows). Stroma composed principally of mature collagenous tissue. 200 ppm 2,3-pentanedione, H&E Original objective magnification: A, 10×; B, 4×; C, 4×; D, 4×; E, 2×; F, 2×. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Figure 3. Luminal Bridging and Secondary Attachment (Epitheliotropism).

Male Wistar Han rats were exposed to 2,3-pentanedione 6 h/day, 5 days/week for 2-weeks. A) Apparent luminal bridging (arrows) of a large bronchus (double asterisk) by intraluminal fibrosis has partially occluded the lumen just proximal to the bifurcation in the lower right (asterisk in lumen of each branch). 200 ppm 2,3-pentanedione followed by 2 week recovery. B) Apparent luminal bridging (arrows) spans the lumen leading from a large bronchus (double asterisk) to one bronchial branch (asterisk). 175 ppm 2,3-pentanedione. C) Small intraluminal fibrous polyp exhibits a narrow base of apparent origin (arrow), and 3 separate foci of secondary attachment to the bronchial mucosal epithelium (arrowheads). 200 ppm 2,3-pentanedione. D) Secondary attachment (arrowhead) of 2 small fibrous polyps. 200 ppm 2,3-pentanedione followed by 2 week recovery. E) Tangled web of 3 interconnected fibrous polyps (arrows), spanning the lumen. 200 ppm 2,3-pentanedione, followed by 2 week recovery. F) Small epithelial bridge (arrow) connecting a large intraluminal fibromyxoid polyp (asterisk) to the adjacent bronchial mucosal epithelium (arrowhead). 200 ppm 2,3-pentanedione. Original objective magnification: A, 2×; B, 2×; C, 20×; D, 10×; E, 10×; F, 10×. H&E. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Figure 4. Intramural Fibrosis.

Male Wistar Han rats were exposed to diacetyl or 2,3-pentanedione 6 h/day, 5 days/week for 2-weeks. A) Intramural (circumferential) fibrosis involving three-fourths of the wall, with partial stenosis of the lumen, epithelial inclusions (arrows), mild inflammation, and partial loss of the smooth muscle layer (between the arrowheads). 175 ppm 2,3-pentanedione. B) Intramural (constrictive) fibrosis, with marked stenosis of lumen, moderate inflammation, and partial loss of the smooth muscle layer (arrowhead). 200 ppm 2,3-pentanedione followed by 2 week recovery. C) Intramural fibrosis in a large bronchus, showing a thick zone of inflamed fibrous tissue (asterisk) with regenerative or atrophic epithelium on the surface (arrowhead). Intramural fibrosis in large bronchi was more frequent than in smaller branches in these studies. 200 ppm diacetyl, followed by 2 week recovery. D) Mixed intramural (arrow) and intraluminal (arrowhead) fibrosis, with stenosis of lumen. Combined lesions of this type were sometimes seen. The fibromyxoid stroma is indicative of an early, developing lesion. 175 ppm 2,3-pentanedione. Original objective magnification: A, 4×; B, 4×; C, 10×; D, 10×. H&E. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Figure 5. Obliterative bronchitis and bronchiolitis.

Male Wistar Han rats were exposed to 2,3-pentanedione 6 h/day, 5 days/week for 2-weeks. A) The lumen of a large bronchial branch is occluded by fibrous tissue, with squamous metaplastic epithelium on the surface (arrowhead). 200 ppm 2,3-pentanedione. B) The lumen of a small bronchial branch is largely occluded by fibrosis (arrow). 200 ppm 2,3-pentanedione; early death (6 days after last exposure). C) The lumen of a small bronchial branch is almost totally occluded by organizing fibrous tissue with fibrin, hemorrhage, remnants of epithelium, and marked inflammation. 150 ppm 2,3-pentanedione. D) The lumen of a small bronchial branch is occluded by fibromyxoid tissue except for a few residual epithelial-lined foci. 150 ppm 2,3-pentanedione. E) The lumen of a small bronchial branch is completely occluded by fibrous tissue, with epithelial remnants at the periphery (arrowheads). 150 ppm 2,3-pentanedione. F) Bronchiolar lumen completely occluded except for small mucosal remnants (arrow). 200 ppm 2,3-pentanedione followed by 2 week recovery. Original objective magnification: A, 4×; B, 4×; C, 10×; D, 10×; E, 10×; F, 10×. H&E. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Figure 6. Inflammation.

Male Wistar Han rats were exposed to diacetyl or 2,3-pentanedione 6 h/day, 5 days/week for 2-weeks. A) Marked lymphocytic inflammation in and around a bronchus, with denuded epithelium. 200 ppm diacetyl. B) Lymphocytic infiltrate in the wall of a terminal bronchiole, with intramural fibrosis (arrow) and regenerative epithelium on the surface (arrowhead). 200 ppm 2,3-pentanedione. C) Marked peribronchial eosinophilic infiltrate. 150 ppm diacetyl followed by 2 week recovery. D) Marked peribronchiolar eosinophilic infiltrate. 150 ppm diacetyl followed by 2 week recovery. Original objective magnification: A, 4×; B, 10×; C, 20×; D, 20×. H&E. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)