Full text of DOI:10.1080/110241500447308

ORIGINAL ARTICLE
Experimental Acute Necrotising Pancreatitis: Evaluation
and Characterisation of a Model of Intraparenchymal
Injection of Sodium Taurocholate in Rats
1
1
2
1
3
1
4
Haim Paran, Ami Mayo, Dvora Kidron, Galit Sivak, Tamar Reshef, Tsahi Vider, Ofer Ziv and
Uri Freund
1
1
2
3
4
From the Departments of Surgery “A” and Pathology, Unit of Clinical Immunology and Unit of Experimental Surgery, Meir
Hospital, affiliated with the Sackler School of Medicine, Tel-Aviv University, Kfar-Sava, Israel
Eur J Surg 2000; 166: 894–898
ABSTRACT
Objectives: To evaluate a simple model that produces progressive dose dependent pancreatitis, by intraparenchymal injection
of sodium taurocholate.
Design: Open laboratory study.
Setting: Teaching hospital, Israel.
Materials: Forty eight Wistar rats.
Interventions: Sodium taurocholate was injected, 0.3 ml/100 g body weight, in concentrations of 5% and 10% into the
pancreatic parenchyma of 32 Wistar rats, resulting in two distinct groups of severity. In 16 sham controls, saline was injected
into the pancreas in similar fashion. Blood samples were withdrawn before, and 6, 24, 48, and 72 hours after induction of
pancreatitis.
Results: Six hours after taurocholate injection, there was a sharp increase in the plasma activities of amylase, lipase, and lactate
dehydrogenase (LDH). After 24 hours plasma activities of amylase and lipase decreased to near normal values while LDH
remained slightly increased for 48 hours and decreased only after 72 hours. At 6 hours after the injection, interleukin-6 (IL-6)
concentrations had increased slightly in the 5% group and decreased to the baseline values at 24 hours. In the 10% group, the
increase in IL-6 values was significantly greater than in the 5% group (p = 0.04), and correlated well with severity of
pancreatitis as defined by histology (p = 0.01) and mortality (p = 0.037). Twenty four hours after injection of taurocholate,
morphological changes comprising diffuse necrosis of the pancreas, fat necrosis, and intestinal dilatation secondary to paralytic
ileus were severe. Histopathological examination of the pancreas showed good correlation with the clinical findings and with
mortality.
No morphological changes were detected when saline was injected into the pancreas (sham control), and only mild rises of
IL-6, lipase, amylase, and LDH activities were seen at 6 hours after injection. The mortality, after 10 days, was 80% in the 10%
taurocholate group, 30% in the 5% taurocholate group, and 0 in the sham control group (p < 0.05).
Conclusion: The intraparenchymal injection of taurocholate is easy to perform and highly reproducible. The histopathological
injury is dose-dependent, as is the mortality. We conclude that this model is valuable for the study of new treatments for
pancreatitis.
Key words: experimental model, pancreatitis, rats, taurocholate.
INTRODUCTION
mental models of acute pancreatitis have been devel-
oped. The more commonly used models are diet-
induced, intravenous or intraperitoneal injections of the
secretagogue caerulein, and retrograde intraductal
injection of sodium taurocholate (1, 10, 17). Most of
these models however, have limitations related to
variability and reproducibility. In 1974 a new and
simple model of experimental pancreatitis was
described (2). A solution of taurocholic acid was
simply injected at several sites in the pancreas.
Although this model has been used in several studies
(5, 8, 9, 14), it has not been adequately evaluated. We
therefore developed and characterised the model of
intraparenchymal injection of sodium taurocholate in
rats.
Acute pancreatitis may present as a mild self-limiting
disorder, or a severe disease which is sometimes fatal.
The pathological features range from mild oedema to
haemorrhage and necrosis. Although its pathophysiol-
ogy has been extensively investigated, it is only partly
understood (3). No specific treatment to prevent the
deleterious effects of the digestive enzymes on the
pancreas and peripancreatic tissues has been found so
far. As there is a definite correlation between the extent
of pancreatic necrosis and the clinical outcome, there is
hope that a specific treatment may be found that would
alter the course of the disease. New treatments are
usually tried in animal models, so numerous experi-

2000 Taylor & Francis. ISSN 1102–4151
Eur J Surg 166

Simple experimental model of pancreatitis 895
Table I. Histopathological score
Oedema:
0
5
0
0
Absent
Diffuse expansion of interlobular septa
Diffuse expansion of interacinar septa
Diffuse expansion of intercellular spaces
1
2
Lobar necrosis in 10 high power fields:
Number of partially necrotic lobes  5
Number of totally necrotic lobes  10
Inflammation:
1
1
0
0
Presence of diffuse leucocyte infiltration in more then 5 high power fields
Presence of micro-abscesses
The histopathological score was calculated by adding the oedema score to the number of partially necrotic lobes multiplied by 5
and the number of totally necrotic lobes multiplied by 10, in 10 high power fields. If there was diffuse leucocyte infiltration in
more then 5 high power fields or micro-abscesses, 10 points were added to each.
MATERIAL AND METHODS
from 20 healthy anaesthetised rats, to establish the
base-line values of all laboratory measurements. To
avoid severe anaemia, the animals in each study group
were randomly divided in two subgroups, and blood
samples were obtained only twice from each animal
during the study. Blood samples (0.8 ml) were with-
drawn at 6 and 48 hours (n = 8), or at 24 and 72 hours
(n = 8) after induction of pancreatitis. The blood was
replaced by an equal volume of normal saline. The
blood samples were immediately centrifuged and the
plasma samples were kept at À70° until tested. The
blood samples were analyzed for pH, PCO2, bicarbo-
nate, base excess (Acid Base Analyzer, ABL30 Radio-
meter), and lactic acid. The sera were assayed for
glucose, electrolytes, and calcium concentrations, and
activities of amylase and lipase (Hitachi Analyzer 737).
IL-6 values (Quantikine M, R&D Systems Minneapo-
lis, USA) were measured by an experienced laboratory
technician (TS) who was unaware of what injection
each rat had had.
Male Wistar rats (300 to 400 g) were kept as outlined in
the “Guide for the Care and use of Laboratory
Animals” (NIH Publication #85–23, 1985). General
anaesthesia was induced by intraperitoneal injection of
6
0 mg/kg of pentobarbitone (Sanosi laboratories, Paris,
France). After anaesthesia, the femoral vein was
cannulated with a 20G polytetrafluoroethylene catheter
(Venflon, Helsingborg, Sweden) which was secured
with a ligature. After each use, the catheter was flushed
with 0.3 ml of heparin solution (1000 units/100 ml
normal saline). The abdomen was then shaved and a
midline laparotomy incision was made. The stomach,
duodenum, pancreas, and spleen were carefully
brought out of the abdominal cavity. Pancreatitis was
induced by multiple (between 7 and 10) intraparench-
ymal injections of a total of 0.3 ml/100 g body weight
of sodium taurocholate solution, at different concen-
trations, through a 30 gauge needle. In sham controls,
saline was injected into the pancreatic parenchyma in a
similar fashion. The injections were made by carefully
introducing the tip of the needle into several parts of the
pancreas, under magnifying lenses, while avoiding
damage to blood vessels and pancreatic ducts. After the
injection, the organs were carefully returned to the
peritoneal cavity, and the incision was closed with a
continuous 3 0 silk suture. The animals were left to
recover for one hour, and put back into unrestraining
cages, 3 rats/cage, and given unlimited access to water
for 24 hours. They were then given unlimited access to
standard rat chow.
Mortality study
Because repeated anaesthesia and blood drawing may
have a biasing effect on mortality, 10 additional rats
from each group cited above, were studied. Pancreatitis
was induced in the same fashion, but without any
further manipulation. They were put in the cages, and
observed every 8 hours for 10 days and the mortality
recorded.
Pathological examination
After the animals had been killed, the pancreas was
harvested and trimmed of fat. Its macroscopic appear-
ance was recorded, and the gland was then fixed in 10%
formaldehyde, processed in paraffin, and stained with
haematoxylin and eosin. Microscopic examination was
made by one pathologist (DK) who was unaware of the
rat’s treatment. The injury was scored by recording the
Study groups: The rats were divided into 3 groups of
16 rats each: sham operated, and saline injected into the
pancreas, 0.3 ml/100 g body weight; 5% taurocholate
solution injected into the pancreas; 10% taurocholate
solution injected in a similar manner.
Biochemical tests: Blood samples were withdrawn
Eur J Surg 166

8
96 H. Paran et al.
number of lobes affected by oedema, partial necrosis,
total necrosis and leucocyte infiltration (Table I). We
also recorded vascular thrombi, bacterial colonies, fat
necrosis, and haemorrhages.
Statistical analysis
Results are expressed as mean (SD). One way analysis
of variance (F test), non-paired t test, chi square test
and ANOVA were used (SPSS, SPSS Inc. software,
USA). Probabilities of less than 0.05 were accepted as
significant.
RESULTS
Taurocholate-induced pancreatitis was characterised
24 hours after induction, by severe morphological
changes in the pancreas including haemorrhages and
necrosis. In addition there was fat necrosis throughout
the peritoneal cavity and pronounced intestinal dilata-
tion. In controls, the injection of saline into the
pancreatic parenchyma was not followed by any
macroscopic damage and all abdominal organs looked
normal.
Six hours after induction of pancreatitis, plasma
activities of amylase, lipase, and LDH were signifi-
cantly increased in both groups. There were also
moderate increases in the control group (Fig. 1). No
consistent significant difference was seen in activities
of lipase and LDH levels between the pancreatitis
groups at 6 hours. Amylase values increased from 1700
(
1
188) to 4213 (637) U/L in the 5% group, and from
830 (157) to 7626 (1666) U/L in the 10% group.
The difference between the groups is significant
p < 0.001). Lipase values increased from 15 (12) to
Fig. 1. Plasma concentrations of amylase, lipase, and lactate
dehydrogenase after induction of pancreatitis. Enzymes
activities are expressed in U/L. Six hours after injection of
taurocholate, activities of all three enzymes had increased
significantly. At 24 hours plasma lipase and amylase had
decreased almost to the baseline values, while lactate
dehydrogenase remained mildly raised up to 72 hours.
Controls had a small rise in the enzymes values probably
related to the mechanical trauma to the gland.
(
1
1
238 (772) U/L in the 5% group and from 16 (10) to
801 (530) U/L in 10% group. The difference between
the groups is significant (p = 0.02). LDH values
increased from 696 (443) to 5347 (2746) U/L in the
5
1
% group and from 670 (541) to 6926 (2572) U/L in the
0% group but there was no significant difference
between the groups. Twenty-four hours after induction
of pancreatitis, serum amylase and lipase activities
decreased to almost baseline values, while LDH values
remained mildly increased up to 48 hours and returned
to baseline after 72 hours.
No consistent significant changes were observed in
plasma concentrations of urea, glucose, or calcium, or
pH in any of the study groups.
There was a mild but significant increase in plasma
lactic acid concentration in the surviving animals at 48
hours and it remained high at 72 hours. This mild
metabolic acidosis was compensated by hyperventila-
tion with a decrease in PCO2 and an unchanged pH.
At 6 hours after induction of pancreatitis, IL-6 values
increased slightly in the controls and the 5% tauro-
cholate group {from 3.9 (4.5) to 42.4 (33.5) pg/L, and
from 4.1 (4.7) to 46.4 (28.5) pg/L, respectively} and
remained considerably raised up to 24 hours {9.7
(2.7) pg/L and 27.5 (37.4) pg/L, respectively}, return-
ing to baseline values at 72 hours. In the 10%
taurocholate group the increase in the IL-6 value was
significantly higher than in the other groups {130.4
(30) pg/L, p < 0.001 in both cases} (Fig. 2).
At 24 hours moderate to severe histopathological
damage was seen in the two groups with pancreatitis,
while in controls, mild oedema was seen in six rats. In
the remaining 10 controls, the pancreas looked normal.
The histological sections of the rats with pancreatitis
showed necrosis and leucocyte infiltration of the
Eur J Surg 166

Simple experimental model of pancreatitis 897
Fig. 2. Two hours after injection of taurocholate, IL-6 values
had increased slighly in control and 5% taurocholate groups,
and remained significantly higher up to 24 hours, but
returned to baseline values at 72 hours. In 10% group the
increase in IL-6 values was significantly greater than in the
other two groups.
Fig. 4. Twenty-four hours after saline injection, the
histological sections of the pancreas showed only mild
perilobular oedema and no necrosis.
DISCUSSION
pancreatic lobes to varying degrees, (Fig. 3 and Fig. 4),
as well as fat necrosis, haemorrhages, and vascular
thrombi in the peripancreatic tissue. Increased con-
centrations of taurocholate resulted in increased mean
damage score which was 3 (2) in the control group, 48
The ideal model of pancreatitis should mimic the
clinical signs, time course, and histopathology of the
human disease. The most commonly used models are
diet-induced, intravenous or intraperitoneal injections
of the secretagogue caerulein, and retrograde intraduc-
tal injection of sodium taurocholate (1, 10, 17). Most of
these models however, have serious limitations related
to technical difficulties, time course of the disease, and
reproducibility. The over-stimulation technique with
caerulein causes only a mild pancreatitis, with no
mortality, and variable slight pathological changes.
Diet-induced models must be used only in mice, with
appreciable technical difficulties, and are almost
invariable lethal. The retrograde influx model, despite
its popularity, has a variable mortality, and the lesion is
confined to the proximal parts of the pancreas. A model
combining retrograde taurocholate injection with sys-
temic caerulein infusion has also been described which
(14) in the 5% taurocholate group and 85 (11) in the
1
0% taurocholate group (p < 0.01). Abundant leuco-
cyte infiltration was associated with higher scores.
The surviving animals were killed after 20 days, and
the pancreas examined. Diffuse scarring was noted,
replacing areas of necrosis, in the taurocholate groups
while the pancreas of the controls looked normal.
Twenty days after induction of pancreatitis, the
surviving animals’ body weight had decreased from
366 (97) to 330 (12) g while the controls’ body weight
remained unchanged at 372 (8) g.
None of the rats in the control group died. In the 5%
group 5 rats died (30%), and in 10% group 13 died
(80%) (p = 0.012) (Fig. 5).
Fig. 5. There was a moderate progressive mortality with
increased concentrations of injected taurocholate. After 10
days, the mortality was 0 in sham controls, 30% in the 5%
taurocholate group, and 80% in the 10% taurocholate group
(p < 0.01).
Fig. 3. Twenty-four hours after injection of taurocholate the
histological sections of the pancreas showed necrosis and fat
necrosis, and the infiltration of leucocytes.
Eur J Surg 166

8
98 H. Paran et al.
correlates well with the human disease. It is dose-
dependent and has good reproducibility, but is time
consuming and quite complicated (16).
pancreatitis: an experimental model. Dig Dis Sci 1974;
9: 1143–1147.
. Becker V. Pathological anatomy and pathogenesis of
acute pancreatitis. World J Surg 1981; 5: 303–313.
. Blamey SL, Imrie CW, O’Neal J, Gilmour WH, Carter
DC. Prognostic factors in acute pancreatitis. Gut 1984;
25: 1340–1346.
. Hadas N, Orda R, Orda S, Bawnik JB, Wiznitzer T.
Experimental pancreatitis in rats caused by intrapar-
enchymal injection of sodium taurocholate. Isr J Med
Sci 1983; 19: 194–197.
6. Heath DL, Cruickshank DH, Gudgeon M, et al. Role of
Interleukin-6 in mediating the acute phase protein
response and potential as an early means of severity
assessment in acute pancreatitis. Pancreas 1993; 66: 41–
1
3
4
We developed a simple model of injections of
sodium taurocholic acid, at different concentrations,
directly into several sites of the pancreatic parenchyma.
The technique is easy, is highly reproducible, and
results in a macroscopically homogeneous injury to the
pancreas, with dose-dependent acinar necrosis and
dose-dependent moderate mortality. This dose-depen-
dent damage is useful as the same model can be used to
study the mild as well as the fatal necrotising form of
the disease, by increasing the concentration of tauro-
cholate, up to the desired degree of damage. The
sodium taurocholate causes a detergent effect, with
diffuse pancreatic necrosis. The early and late histo-
pathological picture mimics the findings in human
disease.
Intraparenchymal injection of sodium taurocholate
resulted in a substantial early increase in plasma
activities of lipase, amylase, and LDH. The increase in
enzyme values seen in the controls probably resulted
from the manual handling and puncture of the gland,
but did not result in any discernible late damage. It is
important to stress, however, that in the clinical
setting, amylase and lipase are not considered to be
of prognostic significance, and are useful only as
markers of the diagnosis of pancreatitis. The increase
in these enzymes comes early in the course of the
pancreatitis and does not correlate with the amount of
damage to the gland or the outcome of the disease
5
4
5.
7
8
9
. Inagaki T, Hoshino M, Hayakawa T, et al. Interleukin 6
is a useful marker for early prediction of the severity of
acute pancreatitis. Pancreas 1997; 14: 1–8.
. Kaplan O, Kaplan D, Kasif E, et al. Effect of delayed
octreotide treatment in acute experimental pancreatitis
in rats. J Surg Res 1996; 62: 109–117.
. Kaplan O, Kushnir T, Sandbank U, Navon G. Acute
pancreatitis in rats: a 31P nuclear magnetic reasonance
study. J Surg Res 1987; 43: 172–178.
0. Lampel M, Kern Hf. Acute interstitial pancreatitis in the
rat induced by excessive doses of a pancreatic
secretagogue. Virchows Arch 1977; 373: 97–117.
1. Moscovitz H, Shofer F, Mignott H, Behrman A,
Kilpatrick L. Plasma cytokine determinations in emer-
gency department patients as a predictor of bacteremia
and infectious diseases severity. Crit Care Med 1994;
1
1
2
2: 1102–1107.
1
2. Norman J, Fink G, Franz M, Carter G. Systemic
cytokine gene expression induced by pancreatitis.
Gastroenterology 1995; 108 (suppl A): 1226.
13. Norman J, Franz M, Riker A, et al. Rapid elevation of
pro-inflammatory cytokines during acute pancreatitis
and their origination within the pancreas. Surg Forum
(4, 15).
The inflammatory cytokine IL-6, on the other hand,
1
994; 45: 145–160.
is generated within the pancreas and its concentration
in the blood is associated with the degree of inflamma-
tion and pancreatic damage (12, 13). Previous studies
have shown that IL-6 values correlated well with the
severity of the disease (6, 7, 11). In the present study,
IL-6 plasma values at 6 hours and 24 hours, correlated
with the concentrations of taurocholate injected. The
two concentrations that we used also correlated with
the pathological score, and with the mortality, much
like the human form of the disease.
1
1
1
1
4. Paran H, Klausner J, Siegal A, et al. Effect of the
somatostatin analogue octreotide on experimental pan-
creatitis in rats. J Surg Res 1996; 62: 201–206.
5. Ranson JHC, Rifkind KM, Turner JW. Prognostic signs
and nonoperative peritoneal lavage in acute pancreatitis.
Surg Gynecol obstet 1976; 143: 209–219.
6. Schmidt J, Rattner DW, Lewandrowski K, et al. A better
model of acute pancreatitis for evaluating therapy. Ann
Surg 1992; 215: 44–56.
7. Steer ML. Workshop on experimental pancreatitis. Dig
Dis Sci 1985; 30: 575–581.
We conclude that this model of experimental
pancreatitis, by intraparenchymal injection in rats, is
simple and highly reproducible. It is suitable for the
investigation of acute pancreatitis, and will be useful in
studies of new methods of treatment.
Submitted August 9, 1999; submitted after revision January
7, 2000; accepted March 8, 2000
1
Address for correspondence:
Haim Paran, M.D.
Department Surgery “A”
Meir Hospital
REFERENCES
1
2
. Aho HJ, Nevalainen TJ, Aho AJ. Experimental pan-
creatitis in the rat. Eur Surg Res 1983; 15: 28–36.
. Bawni JB, Orda R, Wiznitzer T. Acute necrotizing
Kfar-Sava
Israel
Eur J Surg 166