Acinar cell apoptosis and the origin of tubular complexes in caerulein-induced pancreatitis

Abstract

The interrelationship between acinar cell apoptosis and tubular complex formation was examined in caerulein-induced pancreatitis using histology, immunohistochemistry, electron microscopy and DNA gel electrophoresis. Rats were given 8 hourly subcutaneous injections of caerulein, 24 micrograms/kg, for up to 2 days. Morphologically and biochemically typical apoptosis affected 4.6 and 8.9% of acinar cells at 1 and 2 days, respectively, resulting in removal of most acinar cells by 2 days. Consequently, pancreatic ducts, the lining cells expressing bcl-2 and therefore resistant to apoptosis, became much more closely approximated to form the basis of tubular complexes; small numbers of immunohistochemically discrete acinar cells in their lining were either pre-apoptotic resistant to it or newly formed. Proliferation of duct-like lining cells was associated with apoptosis, an increase in islet cells and acinar cell regeneration. There was evidence of duct to acinar cell differentiation but the main increase in acinar cell numbers appeared to derive from proliferation of newly formed acinar cells.

Figure 1

(a) Pancreas of control animal treated with saline/gelatin for 2 days. Arrow, intercalated duct. (b) Pancreas 2 days after commencement of caerulein injections. Tubular complexes are lined by flattened duct-like cells and acinar cells (arrows) are difficult to identify. (c) Pancreas 7 days after commencement of caerulein injections. Acinar cells are once again abundant and readily identifiable. Note mitotic acinar cell (arrow). (d) Apoptotic acinar cells and bodies (arrows) 1 day after commencement of caerulein injections. (H & E; a, b, c, bar = 20 μm; d, bar = 10 μm)

Figure 2

Immunohistochemistry. (a) Pancreas of untreated animal. Ducts are widely separated by closely packed keratin-negative acini. (Cytokeratin). (b) Pancreas 1 day after commencement of caerulein injections. Relatively more tubular complexes. (bcl-2). (c) Pancreas 4 days after commencement of caerulein injections. Tubular complexes with few keratin negative epithelial cells present. (Cytokeratin). (d) Pancreas 7 days after commencement of caerulein injections. Nearly normal. (bcl-2). (e) Pancreas 2 days after commencement of caerulein injections. Note numerous small darkly staining amylase positive apoptotic bodies (dark specks) within epithelium and mainly single residual acinar cells (arrows). (Double staining for amylase (dark) and cytokeratin (light)). (f) Pancreas 4 days after commencement of caerulein injections. Note absence of the amylase positive apoptotic bodies seen in (e) and clustered amylase positive acinar cells, some seen as demilunes surrounding lightly staining complexes lined by duct-like cells (arrows). (Double staining for amylase (dark) and cytokeratin (light)). (a, b, c, d, bar = 50 μm; e, f bar = 25 μm; counterstain haematoxylin).

Figure 3

Immunohistochemistry. Pancreas 4 days after commencement of caerulein injections. Note clustered and mitotic (short arrows) acinar cells (pink). An occasional cell (long arrow) appears cytokeratin (brown) and amylase positive (pink). Islet cell (I) (Double staining for amylase and cytokeratin; bar = 7 μm).

Figure 4

(a) Apoptotic acinar cell 2 days after commencement of caerulein injections. (b) Intraepithelial macrophage (M) containing multiple phagocytosed apoptotic bodies showing varying states of presentation 2 days after commencement of caerulein injections. (c) Small intercellular apoptotic bodies (arrows) in tubular complex of pancreas 4 days after commencement of caerulein injections. Note duct-like cells (D) and single islet cell (I) (d) Tubular complex 2 days after commencement of caerulein injections. Note duct-like (D) and altered acinar cells containing vacuoles (short arrows) and residual bodies (long arrows). (e) Tubular complex 4 days after commencement of caerulein injections. Note duct-like cell (D), islet cell (I), zymogen granules (arrows) and clustered presumably newly forming acinar cells with sparse zymogen granules and absence of vacuoles and residual bodies. (f) Tubular complex 4 days after commencement of caerulein injections. Note developing acinar differentiation in elongated cell with elongated nucleus, broad luminal border, relatively limited RER and sparse small luminal zymogen granules. a, bar = 1.25 μm; b, c, d, bar = 2 μm; e, f, bar = 2.5 μm.

Figure 5

Agarose gel electrophoresis of DNA extracted from saline controls (2 days) (lane 2) and animals 2 days (lane 3) and 7 days (lane 4) after commencement of caerulein injections. Note typical ladder pattern of apoptosis in lane 3. DNA molecular weight markers, lane 1.