Volume 133, Issue 4, Pages 1293-1303 (October 2007) Bacterial Endotoxin: A Trigger Factor for Alcoholic Pancreatitis? Evidence From a Novel, Physiologically Relevant Animal Model  Alain Vonlaufen, Zhihong Xu, Balu Daniel, Rakesh K. Kumar, Romano Pirola, Jeremy Wilson, Minoti V. Apte  Gastroenterology  Volume 133, Issue 4, Pages 1293-1303 (October 2007) DOI: 10.1053/j.gastro.2007.06.062 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Body weights of rats in feeding model. In each of the protocols (single LPS injection and repeated LPS injections) the 4 groups of rats gained weight to a similar extent over the feeding period. Gastroenterology 2007 133, 1293-1303DOI: (10.1053/j.gastro.2007.06.062) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 (A) Effect of ethanol ± single LPS injection on the pancreas. (i) Upper panel: representative H&E-stained pancreatic sections from a control-diet–fed rat and an alcohol-fed rat receiving a single LPS injection (cl and al, respectively). al rats displayed acinar vacuolization (blue arrow), acinar cell necrosis (arrowhead), inflammatory infiltrate (arrow), and hemorrhage (star). Negligible lesions were found in the CL group. Original magnification, 600×. Lower panel: representative H&E-stained pancreatic sections from cl and al animals at higher magnification (1000×). al animals displayed prominent inflammatory infiltration (arrows) compared with cl animals. (ii) Morphometric analysis of acinar vacuolization, necrosis, hemorrhage, inflammatory infiltrate, and edema. Histology scores of al rats were significantly higher than the other 3 groups (control diet + single saline injection, group c; alcohol diet + single saline injection, group a; and control diet + single LPS injection, group cl) (*P < .01 al vs a; #P < .05 al vs cl; n = 7 animals/group). Histology scores were similar in c, a, and cl groups. (B) Effect of ethanol ± repeated LPS injections on the pancreas. (i) Representative H&E-stained pancreatic sections from a control-diet–fed rat and an alcohol-fed rats receiving repeated LPS injections (clr and alr, respectively). alr rats displayed significant acinar cell necrosis (arrows). Negligible lesions were found in c, a, and clr groups. Original magnification, 600×. (ii) Morphometric analysis. Histology scores of alr animals were significantly higher than in the other 3 groups (*P < .001 alr vs c; #P < .01 alr vs a; ‡P < .05 alr vs clr; n = 8 animals/group). Histology scores were similar in the c, a, and clr groups. Gastroenterology 2007 133, 1293-1303DOI: (10.1053/j.gastro.2007.06.062) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 (A) Effect of alcohol ± repeated LPS injections on extracellular matrix deposition in the pancreas. (ii) Representative Masson’s trichrome–stained pancreatic sections from a control-diet–fed rat and an alcohol-fed rat receiving repeated doses of LPS (clr and alr, respectively). Obvious blue fibrotic bands were observed in a periacinar location in the pancreas of alr rats. In addition, the glands showed shrunken acini. In clr animals, blue staining was confined to periductular and perivascular regions, and, notably, acinar size was normal. Original magnification, 1000×. (ii) Morphometric analysis of pancreatic fibrosis. Masson’s trichrome–stained sections of the proximal, middle, and distal pancreas from c, a, clr, and alr animals were assessed by computer-assisted morphometry. alr rats showed a significantly increased fibrosis score compared with the other groups (*P < .001 alr vs c, a, clr; n = 8 animals/group). (B) Effect of alcohol ± repeated LPS injections on collagen deposition in the pancreas. (i) Representative Sirius red–stained pancreatic sections from clr and alr rats. alr animals displayed significant red staining in periacinar areas, indicating collagen deposition. clr rats showed little red staining in periacinar areas and minimal collagen deposition confined to periductular and perivascular regions. (ii) Morphometric analysis of collagen deposition. Sirius red–stained sections of the proximal, middle, and distal pancreas were assessed by computer-assisted morphometry. alr rats showed a significantly increased fibrosis score compared with clr animals (*P < .005; n = 8 animals/group). (C) Effect of alcohol ± repeated LPS injections on fibronectin expression in the pancreas. Representative Western blot and a graph of densitometry results for fibronectin expression in c, a, clr, and alr groups. A significant linear trend for fibronectin expression was observed, with ALr animals showing the highest expression of the protein (*P < .005; n = 8 animals/group). Gastroenterology 2007 133, 1293-1303DOI: (10.1053/j.gastro.2007.06.062) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 (A) In vivo studies: effect of alcohol ± repeated LPS injections on αSMA expression by PSCs. Representative pancreatic sections immunostained for αSMA from a control-diet–fed rat and an alcohol-fed rat receiving repeated doses of LPS are shown (CLr and ALr, respectively). Pancreas of ALr rats displayed marked periacinar αSMA immunoreactivity (indicating activated PSCs). Pancreas of CLr animals showed no αSMA staining. Original magnification, 600×. (B) In vitro studies: effect of LPS on rat PSC activation in vitro. Representative Western blot and densitometry analysis for (i) αSMA and (ii) fibronectin expression by PSCs exposed to culture medium alone (c), LPS .1 μg/mL (L0.1) or LPS 1 μg/mL (L1) for 24 hours, showing a significant increase in αSMA and fibronectin expression, with both LPS concentrations compared with control cells (αSMA: #P < .05 L0.1 vs C; *P < .01 L1 vs C; n = 5 separate PSC preparations; fibronectin: *P < .01 L0.1 and L1 vs C; n = 5 separate PSC preparations). (iii) Synergistic effect of LPS and ethanol on rat PSC activation. Representative Western blot and densitometry analysis of rat PSCs exposed to medium alone (c), ethanol 5 mmol/L (e5), LPS 1 μg/mL (l1), or ethanol 5 mmol/L + LPS 1 μg/mL (e5 + l1), showing a significant linear trend with the highest expression of αSMA seen in PSCs exposed to LPS + ethanol (P < .001; n = 6 separate PSC preparations). Gastroenterology 2007 133, 1293-1303DOI: (10.1053/j.gastro.2007.06.062) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Expression of the LPS receptors TLR4 and CD14 by (A) early culture rat PSCs, (B) culture-activated rat PSCs, and (C) human PSCs, with or without LPS pretreatment. LPS pretreated and untreated rat and human PSCs showed immunoreactivity for TLR4 and CD14. Gastroenterology 2007 133, 1293-1303DOI: (10.1053/j.gastro.2007.06.062) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Induction of TLR4 expression in rat PSCs by LPS. Representative Western blot and densitometry analysis of TLR4 expression in rat PSCs incubated with culture medium alone or LPS 1 μg/mL for 24 hours is shown. Exposure to LPS resulted in a significant induction of TLR4 expression in rat PSCs (*P < .05; n = 3 separate PSC preparations). Gastroenterology 2007 133, 1293-1303DOI: (10.1053/j.gastro.2007.06.062) Copyright © 2007 AGA Institute Terms and Conditions