Journal of Investigative Surgery, 27, 65–72, 2014

Copyright  C 2014 Informa Healthcare USA, Inc.

ISSN: 0894-1939 print / 1521-0553 online

DOI: 10.3109/08941939.2013.837563

ORIGINAL RESEARCH

Expression of Fas (CD95/APO-1) and Fas Ligand (FasL)

in Experimentally-Induced Acute Pancreatitis
Vassilios Pardalis, MD,1 Eleni Palli, MD,1 Maria Lambropoulou, MD, Assistant Professor,2
Christina Tsigalou, MD,3 Stavros Anagnostoulis, MD,1 Grigorios Garoufalis, MD,1
Helen Bolanaki, MD,1 Constantinos Simopoulos, MD, Professor,1
Anastasios J. Karayiannakis, MSc, PhD, MD, Professor1

1
Second Department of Surgery, Democritus University of Thrace, Medical School, Alexandroupolis, Greece, 2 Department
of Histology-Embryology, Democritus University of Thrace, Medical School, Alexandroupolis, Greece, 3 Department of
Microbiology, University General Hospital of Alexandroupolis, Alexandroupolis, Greece

ABSTRACT
Introduction: Acinar cell death is a crucial event in acute pancreatitis (AP) and may occur either by apoptosis or
necrosis. The aim of this study was to investigate the expression of the apoptosis associated proteins Fas and
FasL in experimentally induced severe AP. Methods: AP was induced in 30 rats by injecting 0.2 ml of 4.5% sodium
taurocholate solution into the biliopancreatic duct. Sham operated animals (n = 30) and 10 normal controls were
used for comparisons. Animals were killed at 6, 12, 24, 48, 72 hr and 1 week after operation (five animals at each
time point) and both serum and pancreatic tissue were obtained. The severity of AP was graded by morphological
evaluation and by measuring serum amylase levels. Acinar cell apoptosis was detected by the terminal deoxynu-
cleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Tissue expressions
of Fas and FasL were evaluated by immunohistochemistry. Results: Sodium taurocholate injection resulted in se-
vere acute necrotizing pancreatitis as early as six hr after taurocholate infusion with gradually increasing severity
and a peak at 72 hr, and a significant increase of serum amylase at 6 and 12 hr. Apoptotic acinar cells were ob-
served between 48 and 72 hr. The expression of both Fas and FasL in pancreatic tissue was induced in comparison
with normal controls. Fas expression in AP was higher and statistically significant at 24 hr whereas FasL expres-
sion was consistently lower with a statistical significance observed at 12 hr when compared to sham-operated
animals suggesting Fas upregulation and FasL downregulation in this model of AP. Conclusions: Induction and
sequential changes in the expressions of Fas and FasL occur during taurocholate induced severe AP in rats and
their temporal modulation might associate with acinar cell death by apoptosis.
Keywords: acute pancreatitis, experimental, rat, apoptosis, Fas, FasL

INTRODUCTION severe toxic or physical cellular insult whereas apop-

Development of acute pancreatitis (AP) involves early
acinar cell injury with subsequent recruitment of
inflammatory cells and production of inflammatory
mediators with the extent of acinar cell death and the
degree of the inflammatory reaction being critical de-
terminants of disease severity. Cell death may occur by
two biochemically and morphologically distinct forms
namely apoptosis (also called programmed cell death)
and necrosis [1]. Necrosis occurs under conditions of
tosis is an evolutionarily conserved form of cell suicide
occurring after activation of a constitutively expressed
intrinsic proteolytic mechanism involving a family of
proteases called caspases [2]. There are two pathways
leading to caspase activation; the intrinsic pathway
involving disruption of the mitochondrial membrane
and release of proteins capable of inducing both
caspase-dependent and caspase-independent apopto-
sis and the extrinsic pathway which is mediated by a
group of transmembrane death receptors members of

Received 7 March 2013; accepted 20 August 2013.

Address correspondence to Anastasios J. Karayiannakis, MSc, PhD, MD, Second Department of Surgery, Democritus University of Thrace,
Medical School, 68 100 Alexandroupolis, Greece. E-mail: akarayan@usa.net

65
66 V. Pardalis et al.
the tumor necrosis factor receptor superfamily such as
the CD95 (APO-1/Fas) [3, 4]. Activation of Fas upon
binding of its cognate ligand Fas ligand (FasL) trig-
gers apoptosis by activation of membrane-proximal
(activator) caspases, which in turn, cleave and activate
downstream effector caspases for deployment of the
Fas/FasL apoptotic pathway [5].
Experimental data suggested that acinar cell death
by both apoptosis and necrosis may be involved in
AP with the two mechanisms existing either simul-
taneously or one being predominant over the other
[6–8]. Pancreatic duct ligation-induced pancreatitis in
the opossum and the rat resulted predominantly in
apoptosis in the rat and necrosis in the opossum prob-
ably as a result of different inflammatory cell infiltra-
tions between the two species [9]. An inverse relation-
ship between the severity of pancreatitis and the degree
of acinar cell apoptosis has been noted suggesting that
apoptosis may be a beneficial response to acinar cell in-
jury in AP [10–12].
The aim of this study was to investigate the expres-
sion of the Fas/FasL system and its relationship to
apoptosis in experimental necrotizing AP induced by
sodium taurocholate infusion in the rat.
MATERIALS AND METHODS
Experimental Groups and Procedures
Male Wistar rats weighing between 250 and 300 g
were used in this study. The animals were housed in
a temperature-controlled room (22◦ C) under a 12-hr
light-dark cycle with free access to standard labora-
tory chow and tap water. All animals received humane
care and the procedures were approved by and per-
formed according to the Institutional and Regional Vet-
erinary Office regulations for Animal Care and Use
(Study Protocol No T/2028). The rats were randomly
allocated into three groups as follows: AP (n = 30),
sham-operated (n = 30), and control (n = 10).
AP was induced by pressure controlled intraduc-
tal injection of sodium taurocholate as previously de-
scribed [13, 14]. Briefly, under sevoflurane (3% in oxy-
gen) anesthesia, the abdomen was prepared with a 10%
povidone-iodine solution, a midline laparotomy was
performed under aseptic conditions and the pancreas
was exposed. The biliopancreatic duct was cannulated
at a point just before its opening into the duodenum
and the bile duct was temporarily clamped at the porta
hepatis. Retrograde infusion of 0.2 ml of a 4.5% sodium
taurocholate solution (Sigma, St Louis, MO, USA) into
the pancreatic duct was achieved over one minute un-
der constant pressure of 20 cm H2 O. Sham operated an-
imals received the same volume of 0.9% sodium chlo-
ride. After surgery, the animals were left to recover for
one hour then put back into polycarbonate cages, one
rat per cage, and allowed unlimited access to standard
rat chow and tap water; analgesics, nonsteroidal anti-
inflammatory drugs or antibiotics were not given.
Animals were killed under ether anesthesia at 6, 12,
24, 48, 72 hr and 1 week after the operation (five ani-
mals at each time point). In addition to sham-operated
animals, 10 rats without any intervention were killed
and used as a control group. Blood was collected by di-
rect intracardiac puncture from pancreatitis, sham op-
erated and control animals, and their pancreas was re-
moved, fixed in phosphate buffered formaldehyde (pH
7.4), embedded in paraffin and sectioned at 4 μm for
morphological evaluation and immunohistochemical
studies.
Monitoring and Histological Evaluation
of Acute Pancreatitis
Blood samples collected into plain tubes were allowed
to coagulate and centrifuged at 2,000 g for 10 min.
Serum was separated and analyzed in an automatic an-
alyzer using standard laboratory techniques for deter-
mination of amylase levels.
Changes in pancreatic morphology were evaluated
on hematoxylin-eosin stained slides under light mi-
croscopy. The severity of AP was graded by a detailed
and reliable histological grading system developed by
Spormann et al. [15, 16]. Briefly, interstitial edema, in-
flammatory infiltration, fatty tissue necrosis, acinar cell
necrosis, and hemorrhage were evaluated and graded
by using a 0 to 4 scale. The total score was calculated by
adding the score of each one histopathological variable
separately.
In Situ Apoptosis Detection
The TUNEL (terminal deoxynucleotidyl transferase-
mediated dUTP nick end-labeling) assay was em-
ployed to detect apoptosis in pancreatic tissue by us-
ing the “In Situ Cell Death Detection POD Kit” (Roche
Diagnostics GmbH, Mannheim, Germany) according
to the manufacturer’s instructions. Briefly, paraffin-
embedded tissue samples were deparaffinized, re-
hydrated, incubated with 0.03% H2 O2 to block en-
dogenous peroxidase activity, digested with proteinase
(30 min at room temperature), and the TUNEL-reaction
mixture was applied for 60 min at room temperature.
After washing with phosphate buffered saline (PBS),
sections were incubated with horseradish peroxidase
conjugated streptavidin, developed in diaminoben-
zidine H2 O2 , and counterstained with hematoxylin.
The positive nuclei were counted at high-power field
(×400).
Immunohistochemistry for Fas and FasL
A standard streptavidin-biotin indirect immunoper-
oxidase technique was used for immunostaining.
Journal of Investigative Surgery
 Fas-FasL Expression in Acute Pancreatitis 67

Sections were dewaxed, rehydrated, and incubated
with 0.3% H2 O2 for 20 min to block endogenous perox-
idase. Antigen retrieval was performed by microwave
pretreatment in 0.01 M citrate buffer (pH 6.0) for 15 min
at 700 Watt. After rinsing in 0.01 mol/l of PBS, non-
immune serum was applied for 20 min. to block non-
specific antibody binding. Subsequently, sections were
incubated for 60 min at room temperature with the
primary antibodies. Primary antibodies and dilutions
used were: purified mouse anti-Fas monoclonal (IgG1 )
antibody, 1:200 (BD Transduction Laboratories, San
Diego, CA, USA), and affinity purified rabbit anti-
FasL polyclonal antibody, 1:200 (MBL International,
Woburn, MA, USA). After further rinsing in PBS, sec-
tions were incubated with species-specific biotinylated
secondary antibody (Envision System, Dako, Carpin-
teria, CA., USA) for 60 min at room temperature fol-
lowed by incubation with streptavidin-biotin complex
(Dako) for another 60 min. The peroxidase reaction was
developed with 3,3 -diaminobenzidine tetrahydrochlo-
ride and the slides were counterstained with hema-
toxylin. Formalin-fixed, paraffin-embedded human tis-
sue samples from lymph nodes and tonsils served as
positive controls. Negative controls had the primary
antibody omitted and replaced by PBS.
Evaluation of Immunostaining
The immunostained sections were examined under a
Nikon Eclipse 50i microscope. The expression of Fas
and FasL was estimated semiquantitatively by differ-
ential count of at least 500 cells as follows: negative
(0), <10% of positive cells; weak (+), 10–30% of pos-
itive cells; moderate (++), 30–60% of positive cells;
and strong (+++), >60% of positive cells. Lympho-
cytes present on the same section served as internal
positive controls for Fas or FasL staining. The expres-
sion was defined as membranous when a distinct rim
of immunoreactivity was present on the cell membrane
of most cells, cytoplasmic when the immunoreactivity
was diffusely spread over the cell, and mixed if both
patterns were observed in the same cell or if each pat-
tern was present in a proportion of cells in the same
slide.
Statistical Analysis
All data are presented as means ± standard devia-
tion of the mean (SD). Statistical analysis was per-
formed using the SPSS 18.0 statistical software package
(SPSS Inc., Chicago, IL, USA). Differences within each
group were evaluated by oneway analysis of variance
(ANOVA). Differences between groups were evaluated
by two-way ANOVA followed by an unpaired t test
with Bonferroni correction for multiple comparisons.
Correlations were evaluated using the Pearson corre-
lation coefficient. All tests were two tailed. Statistical
significance was considered when p < .05.
RESULTS
Histology and Severity of Taurocholate
Induced Acute Pancreatitis
Intraductal infusion of sodium taurocholate caused se-
vere changes in pancreatic tissue regarding all vari-
ables studied. These changes resembled hemorrhagic
and necrotizing pancreatitis including expansion of in-
terlobular spaces due to interstitial edema, necrosis of
acinar cells and of peripancreatic fatty tissue, extensive
hemorrhage, and widespread inflammatory cell infil-
tration mostly by neutrophils. Interstitial edema, acinar
cell and fatty tissue necrosis, inflammation, and hemor-
rhage were all evident as early as 6 hr after taurocholate
infusion with increasing severity over time and a peak
at the 72 hr time point (Table 1). The total histological
score of severity showed the same pattern with a grad-
ual increase immediately after induction of pancreati-
tis and a maximum value observed at 72 hr. In contrast,
sham operated animals showed no changes in pancre-
atic morphology apart from occasionally seen leuko-
cyte infiltrates (inflammatory cell infiltration score less
than 1 at all time points).

TABLE 1 Evaluation and grading of particular histological variables and total severity score of sodium taurocholate induced acute
pancreatitis

Time after induction of pancreatitis

Variable 6 hr 12 hr 24 hr 48 hr 72 hr 7 days

Edema 2.60 ± 0.55 3.00 ± 0.71 3.00 ± 0.71 3.00 ± 0.00 4.00 ± 0.00 3.40 ± 0.55
Inflammatory infiltration 2.60 ± 0.55 3.20 ± 0.84 3.40 ± 0.55 3.60 ± 0.55 4.00 ± 0.00 3.40 ± 0.55
Fat necrosis 2.60 ± 0.55 3.00 ± 0.71 3.60 ± 0.55 3.40 ± 0.55 4.00 ± 0.00 3.40 ± 0.55
Acinar cell necrosis 2.80 ± 0.45 3.40 ± 0.89 3.40 ± 0.89 3.80 ± 0.45 4.00 ± 0.00 3.40 ± 0.55
Hemorrhage 2.20 ± 0.45 2.80 ± 0.84 3.00 ± 0.71 3.00 ± 0.00 3.00 ± 0.00 3.40 ± 0.55
Total score 12.80 ± 1.92 15.40 ± 3.71 16.40 ± 2.61 16.80 ± 0.84 19.00 ± 0.00 17.00 ± 2.74

Values are mean (SD) from five animals at each time point.


C 2014 Informa Healthcare USA, Inc.
68 V. Pardalis et al.
FIGURE 1 Serum amylase levels in sodium taurocholate in-
duced acute pancreatitis (AP), sham operated, and control an-
imals. Values are mean (SD) from five animals at each time
point except controls where n = 10 animals. ∗ p < .05 AP versus
sham operated animals at the respective time points.
Serum Amylase Levels
In the AP group, there was a statistically significant in-
crease of serum amylase at 6 and 12 hr after induction
of pancreatitis compared with both the control and the
sham-operated group at the same time points (Figure
1). Serum amylase levels in sham-operated animals at
any time point were not significantly different com-
pared to those of control animals.
Acinar Cell Apoptosis
In AP animals, apoptotic cells (TUNEL-positive nuclei)
were not seen during the 6–24 hr period. Some pos-
itively labeled nuclei appeared 48 hr following pan-
creatitis induction and several were evident at 72 hr
whereas on the 7th day they were scarcely detected.
TUNEL-positive nuclei were selectively located in the
FIGURE 2 Fas (a) and FasL (b) immunoreactivity in normal rat pancreas. Original magnification ×200.
pancreatic acini and were not found in ductal, islet, or
infiltrated cells. There were no positively labeled nuclei
in the pancreata of control or sham operated animals at
any time point.
Immunohistochemical Expression of Fas
and FasL in Pancreatic Tissue
In the normal pancreas, faint Fas and FasL immunore-
activity was seen only occasionally in the cytoplasm of
some acinar and islet cells (Figure 2). Early after induc-
tion of pancreatitis there was induction of both Fas and
FasL expression in pancreatic tissue. Faint to moderate
cytoplasmic Fas and faint membranous and/or cyto-
plasmic FasL immunoreactivity were observed in aci-
nar cells, some ductal cells, and few islet cells (Figures
3 and 4). A similar expression pattern of both Fas and
FasL was also observed in sham-operated animals.
Semiquantitative analysis of Fas/FasL immunoreac-
tivity showed significantly higher Fas expression in AP
animals at all time points compared with control ani-
mals with the highest expression level being observed
at the 24 hr time point (Figure 5). Fas expression was
also induced in sham-operated animals compared to
normal controls achieving statistically significant dif-
ference at 24 hr but was consistently lower when com-
pared with AP animals. The difference between groups
was statistically significant at the 24 hr time point.
Comparison of FasL immunoreactivity among pan-
creatitis and sham-operated animals showed a re-
versed to Fas expression pattern. FasL expression at 12
and 24 hr in both groups was significantly higher com-
pared to normal controls. However, FasL expression
in AP animals was consistently lower when compared
with sham-operated animals the difference being sta-
tistically significant at the 12 hr time point (Figure 6).
There was no significant correlation between Fas
and FasL immunoreactivity either in the AP (r =
0.28, p = .16) or in the sham operation (r = 0.11,
Journal of Investigative Surgery
 Fas-FasL Expression in Acute Pancreatitis 69

FIGURE 3 Immunohistochemical expression of Fas in acute pancreatitis (a–c) and sham-operated (d–f) animals at 24 (a, d), 48
(b, e), and 72 hr (c, f) after operation. Original magnification ×200.

p = .67) group. Fas and FasL immunoreactivity were
not correlated with the score of any histological vari-
able or the total severity score of pancreatitis.
DISCUSSION
In this study, retrograde injection of 4.5% sodium tau-
rocholate solution into the biliopancreatic duct of Wis-

C 2014 Informa Healthcare USA, Inc.
tar rats resulted in severe acute necrotizing pancreatitis
with histological changes culminating 72 hr after tauro-
cholate infusion. The expression of both Fas and FasL
in pancreatic tissue was induced in comparison with
normal controls. Nevertheless, Fas expression in AP
was higher compared to sham-operated animals and
statistically significant at 24 hr whereas FasL expres-
sion was consistently lower with a statistical signifi-
cance observed at 12 hr suggesting Fas upregulation
70 V. Pardalis et al.

FIGURE 4 Immunohistochemical expression of FasL in acute pancreatitis (a–c) and sham-operated (d–f) animals at 12 (a, d), 24
(b, e), and 72 hr (c, f) after operation. Original magnification ×200.

and FasL downregulation in this model of AP. Fas
and FasL expression although minimal, was also ob-
served in sham-operated animals and probably asso-
ciates with a mild inflammatory response to pancreatic
tissue handling and surgical injury per se as evidenced
by the occasional leukocyte infiltrates seen in the pan-
creas of sham-operated animals. Fas and FasL expres-
sion was found in acinar, ductal, and islet cells whereas
TUNEL-positive nuclei detected at 48 and 72 hr were
confined to pancreatic acini only. The small number
of TUNEL-positive nuclei prevented apoptosis quan-
tification and detailed comparisons. There are sev-
eral factors explaining our findings. The discrepancy
of Fas and FasL cellular localization and apoptosis im-
plies that after taurocholate induced pancreatitis apop-
totic cell death involves exclusively acinar cells while
ductal and islet cells might have escaped apoptosis.
The dying cells can be revealed by the TUNEL method

Journal of Investigative Surgery


FIGURE 5 Quantitative evaluation of Fas expression in sodium
taurocholate induced acute pancreatitis (AP), sham operated,
and control animals. Values are mean (SD) from five animals at
each time point except controls where n = 10 animals. ∗ p < .05
AP versus sham operated animals.
for a relatively short time period with positive cells be-
ing those actually undergoing apoptosis and not the
total number of cells died by apoptosis. In addition,
it should be also considered that apoptotic bodies can
disappear and the overall quantification of apoptotic
cell death may be underestimated. In our study, aci-
nar cell apoptosis was minimal whereas both acinar
cell necrosis and total severity score showed the high-
est possible values for this model of acute necrotizing
pancreatitis [15, 16] suggesting that necrosis is the ma-
jor if not the only mode of cell death in this model
of pancreatitis. These findings are in accordance with
the recognized inverse relationship between the sever-
ity of pancreatitis and the extent of apoptosis [9, 10,
12]. Despite minimal acinar cell apoptosis we observed
changes in the expression of Fas and FasL although
no correlation between their expression and severity
of pancreatitis was found. This finding is difficult to
FIGURE 6 Quantitative evaluation of FasL expression in
sodium taurocholate induced acute pancreatitis (AP), sham op-
erated and control animals. Values are mean (SD) from five an-
imals at each time point except controls where n = 10 animals.
∗
p < .05 AP versus sham operated animals.

C 2014 Informa Healthcare USA, Inc.
Fas-FasL Expression in Acute Pancreatitis 71
explain. Fas/FasL induced activation of caspases un-
der certain circumstances such as mitochondrial dys-
function upon ATP depletion may possibly switch the
pattern of acinar cell death from apoptosis to necro-
sis. In addition, caspases have been shown to reduce
acinar cell necrosis and intracellular trypsin activation
suggesting a protective effect in pancreatitis [8]. Early
implication of the Fas/FasL pathway in AP irrespec-
tive of its severity may be a beneficial teleological event
in AP. In this context, prophylactic induction of acinar
cell apoptosis with certain agents has shown to protect
against acinar cell injury in AP [12] whereas inhibition
of apoptosis by cycloheximide increased the extent of
necrosis [11].
In a previous study where pancreatic tissue was ex-
amined 6 hr after infusion of different taurocholate
concentrations (2%, 3.5%, and 5%), the expressions of
Fas and FasL at both mRNA and protein level were
increased after mild/moderate pancreatitis and de-
creased after severe necrotizing pancreatitis [17]. An
increase of soluble Fas and a concomitant decrease of
soluble FasL level were observed in the serum of pa-
tients with AP and these changes were proportional to
the severity of pancreatitis. Furthermore, higher serum
sFas levels and lower serum sFasL levels were both
associated with multiple organ dysfunction syndrome
development and disease related death [18]. Upregu-
lation of both Fas and FasL proportional to the extent
of apoptosis has been also documented in both ani-
mal and human chronic pancreatitis [19, 20]. Tauro-
cholate infusion in rats is a reproducible and reliable
model of severe AP resembling human billiary pancre-
atitis. Although we did not demonstrate a close rela-
tionship between Fas and FasL expression and acinar
cell apoptosis our study revealed a time-dependent im-
plication of the Fas/FasL pathway with early onset af-
ter induction of pancreatitis and preceding culmination
of the inflammatory process. Recruitment of inflam-
matory cells such as neutrophils and lymphocytes af-
ter acinar cell death and production of inflammatory
mediators play a pivotal role in the systemic inflam-
matory reaction and are critical determinants of dis-
ease severity. FasL exhibits a potent chemotactic activ-
ity toward both human and mouse polymorphonuclear
neutrophils thus inducing and/or enhancing inflam-
mation through increased cytokine production [21–24].
The paracrine or autocrine interaction between Fas ex-
pressed on the surface of acinar cell and FasL expressed
by endogenous FasL-positive inflammatory cells or ad-
jacent FasL-positive acinar cells induces Fas-mediated
apoptosis in injured acinar and ductal cells or activated
lymphocytes. Downregulation of FasL may associate
with diminished death of acinar cells and lymphocytes
by Fas/FasL-induced apoptosis and attenuation of tis-
sue destruction by inhibiting FasL-induced inflam-
mation thus providing a mechanism for modulation
of the progression and severity of AP depending on
inflammatory cell type recruitment and activation,
72 V. Pardalis et al.
cellular microenvironment and the noxious agent or
the apoptotic trigger involved. Additional studies con-
cerning the dynamics of severity- and time-dependent
changes of Fas/FasL expression will further our in-
sights about the involvement of the Fas/FasL pathway
in acinar cell death modulation during AP and may of-
fer the basis for potential new therapeutic approaches
as evidenced by some studies where preemptive induc-
tion of apoptosis reduced the severity of AP [11, 25–29].
CONCLUSIONS
In summary, sodium taurocholate injection into the
pancreatic duct of rats induced severe AP with his-
tological changes culminating 72 hr after taurocholate
infusion and apoptotic acinar cell death observed be-
tween 48 and 72 hr. Pancreatic tissue expression of Fas
was significantly induced at 24 hr whereas FasL ex-
pression was significantly reduced at 12 hr compared
to sham-operated animals suggesting Fas upregulation
and FasL downregulation in this model of AP. Tempo-
ral changes in the expressions of Fas and FasL during
AP might associate with acinar cell death by apoptosis.
Declaration of interest: The authors report no conflicts
of interest. The authors alone are responsible for the
content and writing of the article.
REFERENCES
[1] Steller H. Mechanisms and genes of cellular suicide. Science.
1995;267:1445–1449.
[2] Thornberry NA. Caspases: key mediators of apoptosis.
Chem Biol. 1998;5:R97–R103.
[3] Budihardjo I, Oliver H, Lutter M, et al. Biochemical path-
ways of caspase activation during apoptosis. Annu Rev Cell
Dev Biol. 1999;15:269–290.
[4] Walczak H, Krammer PH. The CD95 (APO-1/Fas) and
the TRAIL (APO-2L) apoptosis systems. Exp Cell Res.
2000;256:58–66.
[5] Scaffidi C, Fulda S, Srinivasan A, et al. Two CD95 (APO-
1/Fas) signaling pathways. EMBO J. 1998;17:1675–1687.
[6] Bhatia M. Apoptosis versus necrosis in acute pancreatitis.
Am J Physiol Gastrointest Liver Physiol. 2004;286:G189–G196.
[7] Gukovskaya AS, Pandol SJ. Cell death pathways in pancre-
atitis and pancreatic cancer. Pancreatology. 2004;4:567–86.
[8] Gukovskaya AS, Gukovsky I, Jung Y, et al. Cholecystokinin
induces caspase activation and mitochondrial dysfunction
in pancreatic acinar cells. Roles in cell injury processes of
pancreatitis. J Biol Chem. 2002;277:22595–22604.
[9] Gukovskaya AS, Perkins P, Zaninovic V, et al. Mechanisms
of cell death after pancreatic duct obstruction in the opos-
sum and the rat. Gastroenterology. 1996;110:875–884.
[10] Kaiser AM, Saluja AK, Sengupta A, et al. Relation-
ship between severity, necrosis, and apoptosis in five
models of experimental acute pancreatitis. Am J Physiol.
1995;269:C1295–C1304.
[11] Kaiser AM, Saluja AK, Lu L, et al. Effects of cycloheximide
on pancreatic endonuclease activity, apoptosis, and sever-
ity of acute pancreatitis. Am J Physiol. 1996;271:C982–C993.
[12] Bhatia M. Apoptosis of pancreatic acinar cells in acute pan-
creatitis: is it good or bad? J Cell Mol Med. 2004;8:402–409.
[13] Aho HJ, Koskensalo SM, Nevalainen TJ. Experimen-
tal pancreatitis in the rat. Sodium taurocholate-induced
acute haemorrhagic pancreatitis. Scand J Gastroenterol.
1980;15:411–416.
[14] Aho HJ, Nevalainen TJ, Aho AJ. Experimental pancreati-
tis in the rat. Development of pancreatic necrosis, ischemia
and edema after intraductal sodium taurocholate injection.
Eur Surg Res. 1983;15:28–36.
[15] Spormann H, Sokolowski A, Letko G. Effect of tempo-
rary ischemia upon development and histological pat-
terns of acute pancreatitis in the rat. Pathol Res Pract.
1989;184:507–513.
[16] Spormann H, Sokolowski A, Letko G. Experimental acute
pancreatitis–a quantification of dynamics at enzymic and
histomorphologic levels. Pathol Res Pract. 1989;185:358–362.
[17] Li ZD, Ma QY, Xu J, et al. Colocalized expression of Fas
and Fas-ligand in acute pancreatitis and its correlation with
cell apoptosis. Nan Fang Yi Ke Da Xue Xue Bao (J South Med
Univ). 2006;26:25–29.
[18] Endo S, Inoue Y, Fujino Y, et al. Soluble Fas and soluble
FasL levels in patients with acute pancreatitis. Res Commun
Mol Pathol Pharmacol. 2000;108:179–186.
[19] Su SB, Motoo Y, Xie MJ, et al. Apoptosis in rat spontaneous
chronic pancreatis: role of the Fas and Fas ligand system.
Dig Dis Sci. 2001;46:166–175.
[20] Kornmann M, Ishiwata T, Maruyama H, et al. Coexpression
of FAS and FAS-ligand in chronic pancreatitis: correlation
with apoptosis. Pancreas. 2000;20:123–128.
[21] Ottonello L, Tortolina G, Amelotti M, et al. Soluble Fas lig-
and is chemotactic for human neutrophilic polymorphonu-
clear leukocytes. J Immunol. 1999;162:3601–3606.
[22] Seino K, Iwabuchi K, Kayagaki N, et al. Chemotactic ac-
tivity of soluble Fas ligand against phagocytes. J Immunol.
1998;161:4484–4488.
[23] Miwa K, Asano M, Horai R, et al. Caspase 1-independent
IL-1beta release and inflammation induced by the apopto-
sis inducer Fas ligand. Nat Med. 1998;4:1287–1292.
[24] Yasuda H, Kataoka K, Ichimura H, et al. Cytokine expres-
sion and induction of acinar cell apoptosis after pancre-
atic duct ligation in mice. J Interferon Cytokine Res. 1999;
19:637–644.
[25] Saluja A, Hofbauer B, Yamaguchi Y, et al. Induction
of apoptosis reduces the severity of caerulein-induced
pancreatitis in mice. Biochem Biophys Res Commun. 1996;
220:875–858.
[26] Bhatia M, Wallig MA, Hofbauer B, et al. Induction of
apoptosis in pancreatic acinar cells reduces the sever-
ity of acute pancreatitis. Biochem Biophys Res Commun.
1998;246:476–483.
[27] Hahm KB, Kim JH, You BM, et al. Induction of apop-
tosis with an extract of Artemisia asiatica attenuates the
severity of cerulein-induced pancreatitis in rats. Pancreas.
1998;17:153–157.
[28] Cao Y, Adhikari S, Clément MV, et al. Induction of
apoptosis by crambene protects mice against acute pan-
creatitis via anti-inflammatory pathways. Am J Pathol.
2007;170:1521–1534.
[29] Wang J, Chen G, Gong H, et al. Amelioration of ex-
perimental acute pancreatitis with Dachengqi Decoc-
tion via regulation of necrosis-apoptosis switch in
the pancreatic acinar cell. PLoS ONE 2012;7(7):e40160.
doi:10.1371/journal.pone.0040160
Journal of Investigative Surgery
Copyright of Journal of Investigative Surgery is the property of Taylor & Francis Ltd and its
content may not be copied or emailed to multiple sites or posted to a listserv without the
copyright holder's express written permission. However, users may print, download, or email
articles for individual use.