Massive acinar cell apoptosis with secondary necrosis, origin of ducts in atrophic lobules and failure to regenerate in cyanohydroxybutene pancreatopathy in rats

Abstract

Cyanohydroxybutene (CHB), a glycosinolate breakdown product, causes pancreatic injury when given to animals in large amounts. To determine the course of CHB-induced pancreatopathy, rats were given a single subcutaneous dose of CHB and the pancreas weighed and examined by light and electron microscopy and immunohistochemistry at intervals from 2 h to 28 days. The pancreatic lesion was unusual in that there was marked early oedema with limited inflammatory cell infiltration, rapid synchronous onset of acinar cell apoptosis and early advanced atrophy engendering only a limited regenerative response. Acinar cell apoptosis was atypical in that cell fragmentation was limited and phagocytosis delayed, resulting in extensive secondary necrosis. As ducts were unaffected by CHB, the crowded ducts making up the epithelial component of atrophic lobules could be clearly shown to derive from their condensation and proliferation, not the redifferentiation of pre-existing acinar cells, widely held to produce this lesion. Although the basis of CHB selectivity and toxicity for pancreatic acinar cells remains unknown, the potential therapeutic benefit of such an agent in patients with pancreatitis or pancreatic tumours warrants further investigation.

Figure 1

Body weights of animals as a percentage of starting weight after a single subcutaneous injection of saline (□) or CHB (▪). (n = 4, results expressed as means ± SEM.)

Figure 2

Pancreatic weight as a percentage of body weight in animals after a single subcutaneous injection of saline (□) or CHB (▪). (n = 4, results expressed as means ± SEM.)

Figure 3

Pancreatic morphology after a single subcutaneous injection of saline or CHB. All H & E. (A) 48 h after saline. There is wide separation of ducts (arrows) and islets (I) by closely packed acinar cells (×360). (B) 12 h after CHB. Note numerous apoptotic acinar cells with characteristic nuclear morphology (arrows) (×1280). (C) 18 h after CHB. Most acinar cells contain pyknotic nuclear remnants (arrowheads) and show cytoplasmic swelling and vacuolation; a few appear normal (arrows) (×960). (D) 48 h after CHB. Advanced secondary necrosis affecting all acinar cells in field. Intact duct is indicated by arrow (×380). (E) 96 h after CHB. No acinar cells remain. Atrophic lobules comprise crowded ducts in a connective tissue stroma (×210). (F) 28 days after CHB. Sparse regenerative acini are seen adjacent to islets (arrows). Note few ducts in a collagenous stroma and prominent fatty infiltration (×210).

Figure 4

Pancreatic immunohistochemistry after a single subcutaneous injection of saline or CHB. All with haematoxylin counterstain. (A) 24 h after saline. Widely spaced keratin-positive ducts (arrows) are separated by closely packed keratin negative acinar cells (×170) (B) 48 h after CHB. Widely spaced keratin-positive ducts (arrows) separated by keratin-negative nonviable acinar cells (×190). (C) 96 h after CHB. Lobules comprise crowded keratin-positive ducts separated by loose connective tissue. No acinar cells are seen (×190). (D) 96 h after CHB. Ducts are negative for amylase. Note isolated amylase positive acinar cell (arrow) and periinsular amylase positivity (×170).

Figure 5

Pancreatic ultrastructure after a single subcutaneous injection of CHB. (A) 12 h after CHB. Adjacent apoptotic acinar cells show well-demarcated crescentic clumped chromatin, large nucleolar remnants (arrowhead) and whorling of endoplasmic reticulin (arrows) (×3200). (B) 18 h after CHB. Apoptotic acinar cells show nuclear fragments with crescentic clumped chromatin but dilation of endoplasmic reticulin, swollen mitochondria (arrowheads) and plasma membrane rupture (arrow). Contrast with adjacent viable acinar cells (×3200). (C) 48 h after CHB. Note viable duct epithelial cells (D), residual acinar cell cytoplasmic debris (A) and intra-acinar macrophage (M) laden with residual bodies. The pale cell in the duct epithelium (arrow) is also likely to represent an intraepithelial macrophage (×3700). (D) 96 h after CHB. Duct with typical indented nuclei and sparse organelles of lining cells. Note mitotic lining cell (M), intraepithelial apoptotic body (arrow), and adjacent collapsed redundant basement membrane (arrowhead) (×2650). (E) 96 h after CHB. Activated and mitotic interstitial fibroblasts (×3500). (F) 48 h after CHB. Capillary with intraluminal apoptotic bodies of presumed endothelial cell origin (arrows). Note adjacent intra-acinar macrophage (M) with residual body-laden cytoplasm (×7500).