Original Article / Pancreas
Pathological changes at early stage of
multiple organ injury in a rat model of
severe acute pancreatitis
Xi-Ping Zhang, Jie Zhang, Mei-Li Ma, Yang Cai, Ru-Jun Xu, Qi Xie,
Xin-Ge Jiang and Qian Ye
Hangzhou, China
BACKGROUND: Severe acute pancreatitis (SAP) is a
commonly seen acute abdominal syndrome characterized
by sudden onset, rapid progression and high mortality rate.
The damage in peripheral organs may be more severe than
that in the pancreas, and can even lead to multiple organ
dysfunction. It is critical to recognize early pathological
changes in multiple organs. This study aimed to assess the
early pathological features of damaged organs in a rat model
of SAP.
KEY WORDS: severe acute pancreatitis;
pathological changes;
multiple organs;
injury
Introduction
T
he features of severe acute pancreatitis (SAP) are
fatal pathogenic conditions, rapid progression
METHODS: Thirty clean grade healthy male Sprague-Dawley
and high mortality. It is usually complicated by
rats weighing 250-300 g were randomly divided into a model
syndrome and multiple
control group (n=15) and a sham-operated group (n=15). The systemic inflammatory response
[1, 2]
Current studies have
SAP rat model was induced by sodium taurocholate. Samples organ dysfunction syndrome.
of blood and from multiple organs were collected 3 hours after shown that some inflammatory mediators play an
operation. We assessed the levels of IL-6, TNF-α, PLA2, NO, important role in SAP complicated with multiple organ
ET-1, MDA, amylases and endotoxin in blood and observed injury.[3-5] The pathological changes in multiple organs
the early pathological changes in multiple damaged organs.
and inflammatory mediators in plasma at early stage
RESULTS: Levels of IL-6, TNF-α, PLA2, NO, ET-1 and MDA of SAP were studied in rats, and the causes of multiple
in serum and of amylase and endotoxin in plasma of the organ injury were analyzed. We explored the underlying
model control group rats were significantly higher than those mechanism of SAP, provided a theoretical basis for
of the sham-operated group (P<0.01). Different degrees of
its early pathological changes, and identified relevant
pathological change were observed in multiple damaged
inflammatory mediators.
organs.
CONCLUSION: Multiple organ injury may occur at the early
stage of SAP in rats.
(Hepatobiliary Pancreat Dis Int 2010; 9: 83-87)
Author Affiliations: Department of General Surgery (Zhang XP, Cai Y and
Xie Q), and Department of Pathology (Xu RJ), Hangzhou First People's
Hospital, Hangzhou 310006, China; Zhejiang University of Traditional
Chinese Medicine, Hangzhou 310053, China (Zhang J, Ma ML, Jiang XG
and Ye Q)
Corresponding Author: Xi-Ping Zhang, MD, Department of General
Surgery, Hangzhou First People's Hospital, Hangzhou 310006, China (Tel:
86-571-87065701; Fax: 86-571-87914773; Email: [email protected])
© 2010, Hepatobiliary Pancreat Dis Int. All rights reserved.
Methods
Experimental animals and reagents
Thirty clean grade healthy male Sprague-Dawley rats
weighing 250-300 g were purchased from the Experimental Animal Center of Zhejiang University School of
Medicine (Hangzhou, China).
Sodium taurocholate and sodium pentobarbital
were from Sigma-Aldrich China Inc. (Shanghai, China).
A fully automatic biochemical analyzer was used to
determine the concentration of plasma amylases (U/
L). The kits used were plasma endotoxin tachypleus
amebocyte lysate kit, Shanghai Yihua Medical Science
and Technology Corp. (Institute of Medical Analysis,
Hepatobiliary Pancreat Dis Int，Vol 9，No 1 • February 15，2010 • www.hbpdint.com • 83
Hepatobiliary & Pancreatic Diseases International
Shanghai, China); IL-6 ELISA, pg/ml (ng/L), Shanghai
Shenxiong Biotech Co. (China); TNF-α ELISA, pg/ml
(ng/L), Jingmei Bioengineering Corp. (China); serum
secretory phospholipase A2 enzyme assay ELA (PLA2),
U/ml, R&D Systems Inc. (USA); serum nitrogen oxide
(NO), μmol/L, Nanjing Jiancheng Bioengineering
Research Institute, China; serum endothelin-1 ELA
(ET-1), ng/L (pg/ml), Cayman Chemical Co. (USA;
Catalog Number: 583151); and serum malonaldehyde
(MDA), nmol/ml, Nanjing Jiancheng Bioengineering
Research Institute. All tests were made according to the
instructions with the kits.
tical software (SPSS, Chicago, IL). The Kruskal-Wallis
test or analysis of variance data were recorded as mean±
SD (PLA2 only), the others blood indices were recorded
as M(QR)). All of them were applied for the comparison
of the two groups. Bonferroni's correction was also
applied for comparison. A P value ≤0.05 was considered
statistically significant.
Results
Blood level indices
In the model control group, the levels of IL-6, TNF-α
and NO in serum and the levels of amylases and
Empirical methods and grouping
endotoxin
in plasma were higher than those in the
The improved Aho method[6] was used to induce SAP sham-operated group (P<0.001). In contrast, the levels
in 15 rats.[7, 8] The method of animal model preparation of ET-1 and MDA in the model control group were
was as follows: Rats were anesthetized by intraperitoneal higher than in the sham-operated group (P<0.01). The
injection of 2% sodium pentobarbital (0.25 ml/100 g). level of serum PLA2 in the model group was higher than
In the model control group, we identified the duodenal that in the sham-operated group (P<0.001) (Table).
papilla inside the duodenum duct wall, and then used a
No. 5 needle to drill a hole in the avascular area of the Pathological changes in multiple organs of the
mesentery. A segmental epidural catheter was inserted sham-operated group
into the duodenum cavity through the hole, and
inserted retrogradely into the biliary-pancreatic duct Gross changes
The overall structure of the pancreas remained intact
through the papilla. This was followed by retrograde
and
neither hemorrhage nor evident abnormalities
transfusion of 3.5% sodium taurocholate (0.1 ml/100
g) by a microinjection pump at 0.2 ml/min. The hole in were seen. The color and morphology of the lung were
normal. There were no bleeding points on the lung
the lateral duodenal wall was then sutured.
Another 15 normal rats were randomly assigned to surface or effusions in the thoracic cavity. No swelling
the sham-operated group. We opened the abdominal was evident in the liver, and its color was normal.
cavity in the sham-operated group, turned over the The appearance of the kidney was normal without
pancreas and duodenum, and closed the abdomen. The swelling. Neither bleeding points on the surface of the
rats in all groups were sacrificed by euthanasia 3 hours renal cortex nor visible intestinal dilation, intestinal
wall hyperemia or edema were found. The intestinal
after operation.
After blood was collected from the heart and tissue mucosal surface was smooth, without bleeding and
sample from multiple organs, we observed pathological ulcers. The morphology of thymus tissues, spleen, heart
changes in the pancreas, liver, kidney, terminal ileum, (myocardium), brain and lymph nodes was normal.
lung, spleen, lymph nodes, thymus, heart and brain; and
assessed the levels of IL-6, TNF-α, PLA2, NO, ET-1 and Changes under a light microscope
Pancreas samples showed normal intact gland
MDA in serum, and these of amylase and endotoxin in
structure and mild interstitial edema in a few cases.
plasma.
Neutrophil infiltration was occasional. No acinar
cell, fat necrosis, and hemorrhage were observed. The
Statistical analysis
Statistical analysis was made with SPSS 11.5 statis- structure of lung tissues was normal. Some tissues
Table. Index levels in blood (M(QR) or mean±SD)
Group
IL-6 (ng/L) TNF-α (ng/L) PLA2 (pg/ml) NO (μmol/L) ET-1 (ng/L) MDA (nmol/ml) Amylase (U/L) Endotoxin (EU/ml)
Sham-operated 1.75 (0.65)
3.30 (3.60)
18.70±4.40
10.00 (12.50) 14.05 (1.78) 9.90 (9.90)
2038 (346)
0.02 (0.01)
Model control 4.87 (1.38)* 46.13 (37.95)* 103.69±20.82* 72.50 (17.50)* 17.97 (5.57)# 29.70 (6.60)#
7423 (2275)* 0.04 (0.02)*
*: P<0.001, #: P<0.01, compared to the sham-operated group.
84 • Hepatobiliary Pancreat Dis Int，Vol 9，No 1 • February 15，2010 • www.hbpdint.com
Pathological changes at early stage of multiple organ injury in a rat model of SAP
showed slight edema and inflammatory cell infiltration
of the interstitium and alveolar wall. Complete structure of hepatic lobules and occasional infiltration of
inflammatory cells in the portal area were also found.
Hepatocytes were normal morphologically, but some liver
tissues showed local swelling of hepatocytes, cholestasis
and stenosis of the sinus hepaticus. Renal glomeruli
were normal pathologically in addition to tubules and
interstitium in most rats. Swelling and blurry boundaries
of renal tubular epithelial cells and stenosis of lumens
were found in a few rats. Integrated epidermis and
microvillus structure of intestinal mucosa were seen
without exfoliation, necrosis or edema in the propria layer,
submucosa and placenta percreta in most rats. Thymus
structure was normal, with a clear boundary between
the cortex and medulla, evident lobules, and an intact
envelope. A few epithelial cells with nuclei stained slightly
in the medulla, and a few cells with "vacuoles" were
seen. Mild dilation of the splenic pulp blood sinus, blood
stagnation, and thickened splenic arteriole walls were
seen in a few rats. No pathological changes were found in
the myocardium, brain tissue and lymph nodes in all rats.
In kidney samples, capillary congestion in renal glomeruli,
swelling, scattered necrosis and blurry boundaries in
renal tubule epithelial cells, stenosis or atresia of lumens,
visible protein casts, interstitial edema, inflammatory cell
infiltration and thrombosis were found (Fig. C). There
Pathological changes of multiple organs in the
model control group
Gross changes
Pathological changes were more severe in the
pancreas tail than in the head, and congestion, edema,
hemorrhage and necrosis were evident. Mild liver
swelling was found, and local, gray plaques with obscure
borders occurred in the liver of individual rats. There
were congestion and edema in the pulmonary lobes on
both sides, red bleeding points on the surface of local
pulmonary lobes, and mild effusions in the thoracic
cavity. The color and texture of the spleen were normal
in most rats, but congestion was found in a few. Ileum,
heart, lymph nodes, thymus and brain had no visible
pathological changes.
Changes under a light microscope
In pancreas samples, interstitial congestion and
edema, mild inflammatory cell infiltration, focal
necrosis and mild interstitial hemorrhage were observed.
Local hemorrhage and necrosis became confluent
flakes. There were also capillary congestion, thrombosis
and infiltration of red cells into the interstitium (Fig.
A). In liver tissue, there were hepatocyte swelling,
acidophilic denaturation, concentration of apoptotic
bodies, infiltration of inflammatory cells and leukocyte
adhesion to vessel walls in the portal area, white clots in
venules, dilation and congestion of the sinus hepaticus,
and scattered spotty necrosis in hepatic lobules (Fig. B).
Fig. The multiple organ injury of the model control group at
3 hours (HE, all original magnification ×400). A: pancreas
(massive necrosis of acini); B: liver (lamellar necrosis of hepatic
cells); C: kidney (scattered necrosis in the epithelium of renal
tubules); D: lung (interstitial edema); E : intestinal mucosa
(necrosis and exfoliation of endothelial cells of intestinal villi);
F: myocardium (unclear transverse striations); G: Lymph node
(dilation of lymphatic sinus and sinus cell hyperplasia); H: lymph
node (dilation and spotty necrosis of germinal center); I: thymus
(starry sky change of thymic cortex); J: brain (swelling of cerebral
pyramidal cells and vacuolated nuclei).
Hepatobiliary Pancreat Dis Int，Vol 9，No 1 • February 15，2010 • www.hbpdint.com • 85
Hepatobiliary & Pancreatic Diseases International
were edema in the lung interstitium and alveolar space,
broadened alveolar wall interstitium, inflammatory
cell infiltration, telangiectasis and congestion in the
alveolar wall, and broadened alveolar septa (Fig. D). In
intestinal mucosa, focal necrosis of the ileal mucosa and
inflammatory cell infiltration in various mucosal layers
were found in most rats. Confluence, exfoliation and
defection of villi, broadened intervillous lacunae, parce
arrangement of mucosal glands, decrease of beaker cells
and atrophic mucosa were observed in a few rats (Fig.
E). Focal necrosis of white pulp and lymphoid follicles
and thickened walls of splenic arterioles were found in
the spleen of most rats. There were visible dilation and
congestion of blood sinuses in the red pulp. The spleen
tissue was normal in a few rats. Granulation or lysis of
the cytoplasm of cardiac muscle fibers was found in
individual rats. Mild inflammatory cell infiltration of the
myocardial interstitium was found occasionally. There
was mild inflammatory cell infiltration of the epicardium
in a few rats (Fig. F). Swelling of lymph nodes, dilation
of germinal centers in the nodes and lymphatic sinuses,
and hyperplasia of sinus cells were seen (Fig. G). Spotty
necrosis in the mantle zone and germinal centers of
lymphatic follicles was found in most rats but mild
inflammatory cell infiltration in a few (Fig. H). Mild
histological changes were found in the thymus, with
"starry sky" epithelial cells, fragmentation of nuclei and
decreased lymphocytes. The nuclei of epithelial cells in
the medulla were stained slightly, and vacuolated epithelial
cells occurred in some rats (Fig. I). The brain was normal
pathologically in most rats. Mild swelling of neurons and
brain edema were only found in a few rats (Fig. J).
Discussion
Current studies have confirmed that pancreatic injury
during SAP is complicated by injury of multiple organs
including the liver, lung, kidney, ileum, brain and
heart.[8-10] The pathological changes in these organs
aggravate with time. There have been no reports about
injury of the thymus, lymph nodes and spleen or the
corresponding pathological changes. In this study,
pathological changes were found 3 hours after operation
in multiple organs, mainly edema, necrosis, hemorrhage
and inflammatory cell infiltration. Thrombosis was also
found in the pancreas, liver and kidney, and congestion
was found in the lung and spleen. The multiple organ
injuries occurred in the early stage of SAP in rats. To
understand the severity of multiple organ injury in the
early stage is very important for SAP treatment.
The following are believed to be the underlying
mechanisms of multiple organ injury in SAP: (1)
Various causative agents may cause injury of pancreatic
acinar cells. With the release of pancreatin and activation
of mononuclear macrophages, the excess of neutrophilic
leukocytes may produce or release a great deal of
inflammatory mediators which form a network to cause
inflammatory "cascade effects". This results in multiple
organ injury.[11, 12] (2) These inflammatory mediators
include endotoxin, IL-6, TNF-α, PLA2, NO, ET-1 and
MDA. Endotoxin permeates into blood through damaged
intestinal mucosa at early stages. The mononuclear
phagocyte system is activated by endotoxin in blood and
initiates an inflammatory reaction, which aggravates
the injury of the intestinal mucosal epithelium, inhibits
the proliferation of intestinal endothelial cells and
further delays recovery from SAP. Therefore, this causes
endotoxin and cytokines to form a vicious cycle.[13, 14]
We suggest that endotoxin plays an important role in
promoting SAP during the progression of multiple organ
injury. As one of the important cytokines participating
in the pathogenesis of SAP,[15] TNF-α, a primary proinflammatory factor, directly injures the cells of multiple
organs, and causes ischemia, hemorrhage, necrosis,
inflammation and edema. As the secondary chemotactic
factor of inflammatory factors, TNF-α initiates a
cascade reaction, accumulates neutrophilic leucocytes,
increases ICAM and VCAM levels, and stimulates the
production of NO, ROS and other pro-inflammatory
factors such as IL-6 and IL-1β.[16] IL-6 has extensive proinflammatory effects to cause tissue damage. IL-6 also
influences the coagulation and fibrinolytic systems, and
causes thrombosis of fibrin in blood vessels.[17] PLA2
is an important mediator of multiple organ injury.
When SAP occurs, a great amount of PLA2 is released
by polymorphonuclear leukocytes and mononuclear
macrophages, stimulated by endotoxin. This enters
the blood, attacks and degrades phospholipids in
membranes, damages membrane stability and causes
massive leakage of lysosomal enzymes. On the other
hand, PLA2 generates bioactive free fatty acids and
lytic lecithin to destroy the functions and structures of
cells and organs.[18, 19] As a final common inflammatory
mediator of the cascade reaction in the inflammatory
reaction,[20] NO is regarded as an index for predicting
the pathological severity of SAP. Some researchers
believe that a low concentration of endogenous NO
protects against ischemic reperfusion injury, prevents
the increase of intestinal vasopermeability, and obviates
endotoxemia and bacteria translocation. MDA is a stable
metabolite of oxygen free radicals. Because oxygen free
radicals attack biological structures and biochemical
compounds, MDA indirectly reflects the severity of
their effects. ET causes continuous spasm of pancreatic
86 • Hepatobiliary Pancreat Dis Int，Vol 9，No 1 • February 15，2010 • www.hbpdint.com
Pathological changes at early stage of multiple organ injury in a rat model of SAP
556.
8 Zhang XP, Zhang L, Wang Y, Cheng QH, Wang JM, Cai W,
et al. Study of the protective effects of dexamethasone on
multiple organ injury in rats with severe acute pancreatitis.
JOP 2007;8:400-412.
9 Zhang XP, Tian H, Wu DJ, Feng GH, Chen L, Zhang J, et
al. Pathological changes in multiple organs of rats with
severe acute pancreatitis treated by baicalin and octreotide.
Hepatobiliary Pancreat Dis Int 2009;8:85-92.
10 X
u GF, Lu Z, Gao J, Li ZS, Gong YF. Effect of ecoimmunonutrition supports on maintenance of integrity of intestinal
mucosal barrier in severe acute pancreatitis in dogs. Chin
Med J (Engl) 2006;119:656-661.
11 Kimura Y, Hirota M, Okabe A, Inoue K, Kuwata K, Ohmuraya
M, et al. Dynamic aspects of granulocyte activation in rat
severe acute pancreatitis. Pancreas 2003; 27:127-132.
12 M
ikami Y, Takeda K, Shibuya K, Qiu-Feng H, Egawa S,
Sunamura M, et al. Peritoneal inflammatory cells in acute
pancreatitis: Relationship of infiltration dynamics and
Funding: This study was supported by grants from the Intensive
cytokine production with severity of illness. Surgery 2002;
Foundation Project for Technology of Hangzhou (2004Z006).
132:86-92.
Ethical approval: This study was approved by the Ethics 13 R
ahman SH, Ammori BJ, Holmfield J, Larvin M, McMahon
Committee of the hospital.
MJ. Intestinal hypoperfusion contributes to gut barrier
Contributors: ZXP, ZJ and MML wrote the first draft. All
failure in severe acute pancreatitis. J Gastrointest Surg 2003;
authors contributed to the intellectual context and approved the
7:26-36.
final version. ZXP is the guarantor.
14 A mmori BJ, Fitzgerald P, Hawkey P, McMahon MJ. The early
Competing interest: No benefits in any form have been received
increase in intestinal permeability and systemic endotoxin
or will be received from a commercial party related directly or
exposure in patients with severe acute pancreatitis is not
indirectly to the subject of this article.
associated with systemic bacterial translocation: molecular
investigation of microbial DNA in the blood. Pancreas 2003;
26:18-22.
15 Zhao YF, Zhai WL, Zhang SJ, Chen XP. Protection effect of
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Received May 12, 2009
severe acute pancreatitis. World J Gastroenterol 2007;13:548Accepted after revision August 20, 2009
capillaries and promotes the inflow of calcium ions
to directly injure pancreatic cells. ET also causes
contraction of coronary arteries and influences heart
function to injure pancreatic tissue indirectly.[21]
As shown in the experimental results, the levels
of inflammatory mediators were higher in the model
control than in the sham-operated group in this study.
Inflammatory cell infiltration was found in the pancreas,
liver, kidney, lung, ileum, heart, lymph nodes and
other tissues, and pathological changes such as edema,
hemorrhage and necrosis were found in multiple organs
of the model control group. All these changes indicate
that the interaction of inflammatory mediators causes
the pathological changes in multiple organs at the early
stage of SAP.
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