Research Article
Cholecystokinin-8 Treatment of Pigs with Induced Acute Pancreatitis Significantly Reduces Acinar Necrosis and Edema of Pancreatic Tissue
Katharina Grupp1*, Sarah Bonk1, Annika Poppe2, Lena Seifert1, Karin Wodack2, Constantin Trepte2, Matthias Reeh1, Andreas Gocht3, Oliver Mann1, Jakob R Izbicki1 and Kai Bachmann1
*Corresponding author: Katharina Grupp, Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg- Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany
Published: 20 Jun, 2018
Cite this article as: Grupp K, Bonk S, Poppe A, Seifert
L, Wodack K, Trepte C, et al.
Cholecystokinin-8 Treatment of Pigs
with Induced Acute Pancreatitis
Significantly Reduces Acinar Necrosis
and Edema of Pancreatic Tissue. Clin
Surg. 2018; 3: 1988.
Abstract
Objective: Acute pancreatitis is an inflammatory process of the pancreas and a leading cause of
hospitalization amongst gastrointestinal disorders. Previously, cholecystokinin (CCK) has been
described to play a role in regeneration of pancreas. This study was undertaken to get more insights
in the function of cholecystokinin octapeptide (CCK - 8) during induced pancreatitis in an animal
model.
Methods: In this study, acute pancreatitis was induced in 38 pigs. Two hours after the induction
of acute pancreatitis, the animals were grouped according to the melatonin treatment into the
following two groups: group 1/CCK - 8 group and group 2/non - CCK - 8 groups. Intraoperative
clinical data, postoperative blood parameters and ‘Porcine Well-Being’ (PWB) score, as well as post
- mortal histopathological data were analysed.
Results: At baseline, physiologically parameters of the pigs of both groups were comparable. No
differences were observed regarding the overall survival of animals (p=0.97). Postoperative PWB
score were significantly better in animals treated with CCK - 8 as compared to the control group
(p=0.029). Moreover, histopathological analysis of the pancreatic tissue revealed that acinar necrosis
and edema were significant reduced in the CCK - 8 group in comparison to the control group
(p=0.016 and p=0.019).
Conclusion: CCK - 8 treatments reduces acinar necrosis and edema of pancreatic tissue after
induction of an acute pancreatitis in pigs. Thus, it can be speculated that CCK - 8 may be useful as a
therapeutic medical treatment of severe acute pancreatitis.
Keywords: Acute pancreatitis; Experimental model; Cholecystokinin - 8; CCK-8
Introduction
Acute pancreatitis is the leading gastrointestinal cause of hospitalization. In the majority of cases, acute pancreatitis manifests as a mild, self-limited course. However, in 15% to 25% of patients, it leads to tissue necrosis and infection with severe complications including endocrine and exocrine pancreatic insufficiency, organ failure, fistulae, bleeding, and death [1]. The gastrointestinal peptide cholecystokinin (CCK) acts physiologically on CCK receptors initiating various intracellular signaling pathways, which in turn result in enzyme/acid secretion, cellular proliferation and antiapoptosis, and cell migration [2-6]. Intracellular signaling pathways activated involve the hydrolysis of phosphatidylinositol bisphosphate by phospholipase C to generate inositol trisphosphate and diacylglycerol, which subsequently induce calcium mobilization and activation of protein kinase C [7]. Several of these pathways involve activation or cross talk with tyrosine kinase receptors and proliferative pathways associated with cell growth including mammalian target of rapamycin, Akt, and extracellular signal-regulated kinases [2]. In addition, CCK has been shown to play an important role in regulating pancreatic growth in animals and pancreatic regeneration [8-11]. This study was undertaken to get more insights in the clinical effect of CCK - 8 treatments in pigs after induced acute pancreatitis. Our data demonstrate that CCK - 8 reduces acinar necrosis and edema of pancreatitis tissue in pigs. Thus, it can be speculated that CCK - 8 may be useful as a therapeutic treatment of patients with severe acute pancreatitis.
Table 1
Table 2
Table 3
Research Design and Methods
Study design
The study was approved by the Governmental Commission on
the Care and Use of Animals of the City of Hamburg. The animals
received care in compliance with the “Guide for the Care and Use of
Laboratory Animals” (NIH publication No. 86 - 23, revised 1996).
38 pigs (German Hybrid) were included and randomized to two
different treatment groups: group 1 (CCK - 8, n=18) and group 2
(non - CCK - 8; control group; n=20). Acute pancreatitis was induced
in both groups but only the animals of the CCK – 8 - groups were
treated with CCK - 8.
Surgical preparation
After fasting overnight with free access to water, ketamine (10 mg/
kg), midazolam (0.5 mg/kg), azaperone (4 mg/kg) and atropine (0.0015
mg/kg) were administered for premedication. For monitoring of heart
rate and oxygen saturation a 5-lead electrocardiogram and pulsed
oximetry were used. After preoxygenation anaesthesia was induced
by intravenous injection of 0.5 mg/kg midazolam. The animals were
intubated and ventilated in a pressure - controlled mode assuring tidal
volumes of 8 -12 ml/kg and an endexperiatory pCO2 of 35 mmHg to
40 mmHg using an inspiratory oxygen concentration of 0.35 (Zeus,
Draeger Medical Systems, Lübeck, Germany). Continuous infusion
of fentanyl (0.05 mg/kg/h) and sevoflurane (Fet 2.0) was used for
balanced anaesthesia. After cleaning, shaving, disinfection and sterile
draping, the femoral artery was cannulated using a 5 F thermistor
tipped arterial catheter (PICCO, PV 2015L20, Pulsion, Germany)) for
advanced hemodynamic monitoring. Two central venous catheters
were surgically introduced into the internal and external jugular vein
for volume administration and injection of cold indicator for transcardiopulmonary
thermodilution using a PiCCO plus monitoring
system (version 6.0, Pulsion Medical Systems, Munich, Germany).
Fluid management was identical for all animals. A basal infusion rate
of 13 ml/kgBW/h was administered using hydroxyethyl starch 6%
130/0.4 and Ringer’s solution at a fixed ratio of 1:2. Macrocirculation
was assessed continuously and maintained identically in all animals
during the entire procedure according to an established algorithm
for goal-directed fluid management [12-14]. Body temperature was
kept constant between 38°C to 39°C using forced-air warming and
a heating pad. After repositioning of the pigs into supine position, a
gastric tube was placed and the abdomen was opened by a transverse
upper laparotomy. A urinary catheter was placed directly into the
bladder for urinary drainage. The pancreas and duodenum were
mobilized and fixed at the laparotomy incision for intraoperative
measurements. After dissection and cannulation of the main pancreatic
duct (Vasofix 0,8 mm, B. Braun, Melsungen, Germany) between
pancreas and duodenal wall a flexible polarographic measuring
probe (CCP1, Licox, Kiel, Germany) for continuous measurement of
the tissue oxygen tension (tpO2) was placed in the pancreatic head
[15,16]. After a few minutes of equilibration the baseline values of
all parameters (M0) were measured. According to the protocol the
measurements includes blood gas analysis, measurement of tissue
oxygenation (tpO2) and the microcirculation in the pancreatic head
with a laser Doppler imager (LDI, Moore, UK) [17]. Afterwards acute
necrotizing pancreatitis was introduced by intraductal infusion of
glycodeoxycholic acid (GDOC, 10 mmol/l, pH8, Sigma - Aldrich, St.
Louis, MO, USA) over a period of 15 min as previously described,
using an automated infusion system (Perfusor® fm (MFC), B Braun,
Melsungen, Germany) to avoid pancreatic pressure necrosis [17-19].
The cannula was removed and the pancreatic duct was ligated. 60
minutes (M1) and 120 (M2) min after completion of the intraductal
infusion measurements were repeated. Directly after M2, the animals
of group 1 (CCK treatment) received a bolus of 0,5 μg/kg KG CCK - 8
(CCK8, Sigma - Aldrich, Chemie GmbH, Germany) was applied via
the central venous catheter. After start of the therapy a stabilization
period of 30 min was allowed before the effects were measured every
60 min (M3 - 8). After the last intraoperative measurement (M8) all
catheters were removed except the central venous catheter, that was
subcutaneously tunneled to the dorsal neck of the pig for application
of analgesic medication and blood gas testing in the postoperative
course. The abdominal cavity and incision of the neck were closed
and anesthesia was terminated. The animals were extubated and if
sufficient spontaneous breathing was assured, they were transferred
to heated boxes in the animal facility. For 7 days the animals were
closely monitored and analgesics were given every 4 hr to 6 hr
(piritamide 15 mg, equivalent to 10 mg morphine). Once a day blood
samples and blood gas analysis were performed and the animals were
evaluated for their fitness using two scores that had been used earlier
by our group [20]. Animals surviving the observation period were
re-anesthetized on the 7th postoperative day, and sacrificed by fast
injection of potassium chloride during anesthesia. The pancreas was
removed for histopathologic examination and molecular biological
analysis. In animals that died during the postoperative course the
pancreas was removed directly after death. Representative specimens
of the pancreas were taken. Parts of each pancreatic area, that is, head,
corpus, and tail were stored in 3.5% buffered formalin, separately. The
tissues were then processed, embedded in paraffin and 5 μm slices
were stained with hematoxylin and eosin. The slices were examined by
an experienced pathologist. Specimens were examined by a treatment
- blinded experienced pathologist. The histopathologic evaluation
of the pancreatic lesions based on a previous publication [21].
Histopathologic changes were evaluated for each pancreatic area, that
is, head, corpus, and tail, separately, and for each anatomic region a
total score ranging from 0 (no alterations) to 12 (severe pancreatitis)
was determined (Table 6).
Statistical analysis
Statistical analysis was performed with SPSS® for Windows®
(Version 22.0) (SPSS Inc., Chicago, IL). Descriptive analysis of
parametric parameters is expressed as means and standard deviation.
Ordinal data were expressed as median and range. For analysis of the
difference between the groups in repeated measurements the variance
analysis for repeated measurements (ANOVA) followed by a time -
by - treatment -interaction test was used. Additionally, the area under
curve was calculated during the intraoperative treatment (M2 to M8).
Differences between the treatment groups were analysed using oneway
ANOVA. Significance statements refer to p values of two-tailed
tests that were less than 0.05.
Table 4
Table 5
Table 6
Figure 1
Figure 2
Results
Baseline characteristics
The animals were grouped according to the operative procedure
into the following two groups: group 1/CCK - 8 group and group 2/
non – CCK - 8 groups. A total of 18 animals were treated with CCK
- 8, while a total of 20 animals were grouped to the control cohort.
At baseline, the clinical characteristics of the animals of both groups
were similar as demonstrated in Table 1. In detail, the mean length
and weight were 98.4 cm and 30.2 kg of the CCK - 8 group and 98.3
cm and 30.7 kg of the control group 98.3 (p=0.95 and p=0.62).
Overall survival
No differences were observed regarding the overall survival of the
pigs (CCK-8 group: 153h versus non-CCK-8 group: 144h; p=0.97) as
demonstrated in (Table 1) and (Figure 1).
Tissue oxygenation of the pancreatic tissue
(Figure 2) and (Table 2) show the tissue oxygenation of the
pancreas during the operative course of both groups (M0-M8).
The oxygenation data were comparable in both analyzed groups
(p=0.547).
Intraoperative hemodynamic data and blood test results
All animals were kept in stable hemodynamic conditions during
the operation. The hemodynamic data are shown in Table 3 and
the results of the blood tests in (Table 4). These data demonstrate
that there were no significant differences concerning the analysed
parameters of both animal cohorts.
Postoperative fitness and PWB score of the animals
As shown in (Table 5), there were no significant differences
regarding the fitness score of animals which were treated with CCK
- 8 compared to the animals of the control group (p=0.093), only at
M10 significant advantages were detected. Interestingly, pigs of the
CCK - 8 group had an increased PWB score as compared to the pigs
of the control group (p=0.029). In detail, significant advantages were
present at M10, M11 and M12.
Histopathological analysis
The histopathological analysis revealed that acinar necrosis and
edema were significant reduced in the CCK - 8 group as compared to
control group (p=0.016 and p=0.019) as demonstrated in (Table 6).
The overall score showed a tendency to favourable results in the CCK
- 8 group but missed statistical significance (p=0.062).
Discussion
CCK has been described to play a role in regeneration of pancreas. This study was undertaken to get more insights in the function of CCK - 8 during induced pancreatitis in an animal model. In summary, our data demonstrate that the treatment with CCK - 8 reduces acinar necrosis and edema of the pancreatic tissue and reduce the severity of the disease after experimental induction of acute pancreatitis. The majority of acute pancreatitis is mild and associated with a short time of hospitalization [1]. This mild form of acute pancreatitis is characterized by the absence of organ failure and/or pancreatic necrosis, while the severe form is associated with a systemic inflammatory response syndrome and/or organ failure [22]. The presence of organ failure and infected pancreatic necrosis is strongly correlated with the prognosis of patients [23]. Our data demonstrated that the overall survival rate was comparable in both analysed subgroups of pigs after the induction of acute pancreatitis. However, we observed an increased PWB score in pigs, which were treated with CCK - 8 as compared to the pigs of the control group. Previously, Jia et al. [24] had demonstrated that the most favorable strategy for the treatment of acute pancreatitis is to maintain the pancreas at rest during an early stage for only a short period, followed by pancreatic stimulation. Thus, it can be speculated that the treatment of the animals with CCK - 8 enhanced the recovery of pancreatic function. Pathophysiologically, inappropriate activation of pancreatic proenzymes within the gland itself leads to tissue and microvascular injury, release of pro-inflammatory mediators, and local inflammation [1]. During earlier stages of acute pancreatitis pro-inflammatory cytokines such as tumor necrosis factor α are produced by the pancreatic acinar cells and Interleucin - 6 and -10 are expressed on the cells surface [25-27]. Moreover, anti - inflammatory cytokines are produced to inhibit the immune response, rendering the host at risk for systemic infection [28]. Interestingly, in our study the treatment of the animals with CCK - 8 was linked to a reduction of acinar necrosis and edema of the pancreatic tissue. Previously, Elsässer et al. [11] had demonstrated that CCK plays an important role in regulating pancreatic regeneration. Our data underline the assumption that CCK - 8 may have a positive effect on the recovery of the pancreas after the induction of an acute pancreatitis. However, the underlying biological mechanism remains elusive. An issue, worth to be discussed is the interval between induction of pancreatitis and beginning of treatment. In our study, the interval chosen was rather short. However to our understanding this seems to be adequate, because the direct intraductal injection of bile acid induces an acute pancreatitis within a few minutes, which is much faster than acute biliary pancreatitis found in the clinical situation [29,30]. In our experimental setting a severe acute pancreatitis was observed macroscopically in all animals prior to beginning of therapeutic intervention. If the interval between induction and beginning of the treatment is too long, the effect of improvement of the pancreatic microcirculation may fail to appear when fulminate necrosis are already present, as the rationale for the treatment approach is to improve microcirculatory perfusion and thereby save not yet irreversible injured tissue from infarction and necrosis [31,32]. In summary, we demonstrated that CCK-8 reduces acinar necrosis and edema of pancreatic tissue after induction of an acute pancreatitis. Thus, it can be speculated that CCK-8 may be useful as a therapeutic medical treatment of severe acute pancreatitis.
References
- Bendersky VA, Mallipeddi MK, Perez A, Pappas TN. Necrotizing pancreatitis: challenges and solutions. Clin Exp Gastroenterol. 2016;9:345-50.
- Smith JP, Solomon TE. Cholecystokinin and pancreatic cancer: the chicken or the egg? Am J Physiol Gastrointest Liver Physiol. 2014;306(2):G91-101.
- Dembinski AB, Johnson LR. Stimulation of pancreatic growth by secretin, caerulein, and pentagastrin. Endocrinology. 1980;106(1):323-8.
- Mainz DL, Black O, Webster PD. Hormonal control of pancreatic growth. J Clin Invest. 1973;52(9):2300-4.
- Dockray GJ, Moore A, Varro A, Pritchard DM. Gastrin receptor pharmacology. Curr Gastroenterol Rep. 2012;14(6):453-9.
- Grabowska AM, Watson SA. Role of gastrin peptides in carcinogenesis. Cancer Lett. 2007;257(1):1-15.
- Dufresne M, Seva C, Fourmy D. Cholecystokinin and gastrin receptors. Physiol Rev. 2006;86(3):805-47.
- Solomon TE, Petersen H, Elashoff J, Grossman MI. Interaction of caerulein and secretin on pancreatic size and composition in rat. Am J Physiol. 1978;235(6):E714-9.
- Solomon TE, Vanier M, Morisset J. Cell site and time course of DNA synthesis in pancreas after caerulein and secretin. Am J Physiol. 1983;245(1):G99-105.
- Zucker KA, Adrian TE, Bilchik AJ, Modlin IM. Effects of the CCK receptor antagonist L364,718 on pancreatic growth in adult and developing animals. Am J Physiol. 1989;257(4 Pt 1):G511-6.
- Elsässer HP, Adler G, Kern HF. Time course and cellular source of pancreatic regeneration following acute pancreatitis in the rat. Pancreas. 1986;1(5):421-9.
- Kubitz JC, Forkl S, Annecke T, Kronas N, Goetz AE, Reuter DA. Systolic pressure variation and pulse pressure variation during modifications of arterial pressure. Intensive Care Med. 2008;34(8):1520-4.
- Reuter DA, Bayerlein J, Goepfert MSG, Weis FC, Kilger E, Lamm P, et al. Influence of tidal volume on left ventricular stroke volume variation measured by pulse contour analysis in mechanically ventilated patients. Intensive Care Med. 2003;29(3):476-80.
- Kubitz JC, Annecke T, Forkl S, Kemming GI, Kronas N, Goetz AE, et al. Validation of pulse contour derived stroke volume variation during modifications of cardiac afterload. Br J Anaesth. 2007;98(5):591-7.
- Lee SK, Morabito D, Hemphill JC, Erickson V, Holcroft JJ, Derugin N, et al. Small-volume resuscitation with HBOC-201: effects on cardiovascular parameters and brain tissue oxygen tension in an out-of-hospital model of hemorrhage in swine. Acad Emerg Med. 2002;9:969-76.
- Boekstegers P, Weiss M. Tissue oxygen partial pressure distribution within the human skeletal muscle during hypercapnia. Adv Exp Med Biol. 1990;277:525-31.
- Freitag M, Standl TG, Kleinhans H, Gottschalk A, Mann O, Rempf C, et al. Improvement of impaired microcirculation and tissue oxygenation by hemodilution with hydroxyethyl starch plus cell-free hemoglobin in acute porcine pancreatitis. Pancreatology. 2006;6(3):232-9.
- Kusterer K, Poschmann T, Friedemann A, Enghofer M, Zendler S, Usadel KH. Arterial constriction, ischemia-reperfusion, and leukocyte adherence in acute pancreatitis. Am J Physiol. 1993;265(1):G165-71.
- Williams JA. Regulation of pancreatic acinar cell function. Curr Opin Gastroenterol. 2006;22(5):498-504.
- Toouli J, Brooke-Smith M, Bassi C, Carr-Locke D, Telford J, Freeny P, et al. Guidelines for the management of acute pancreatitis. J Gastroenterol Hepatol. 2002;17 Suppl:S15-39.
- Wodack KH, Poppe AM, Tomkötter L, Bachmann KA, Strobel CM, Bonk S, et al. Individualized early goal-directed therapy in systemic inflammation: is full utilization of preload reserve the optimal strategy? Crit Care Med. 2014;42(12):e741-51.
- Tenner S, Baillie J, DeWitt J, Vege SS. American College of Gastroenterology guideline: Management of acute pancreatitis. Am J Gastroenterol. 2013;108(9):1400-15.
- Petrov MS, Shanbhag S, Chakraborty M, Phillips ARJ, Windsor JA. Organ failure and infection of pancreatic necrosis as determinants of mortality in patients with acute pancreatitis. Gastroenterology. 2010;139(3):813-20.
- Jia D, Yamamoto M, Otsuki M. Effect of endogenous cholecystokinin on the course of acute pancreatitis in rats. World J Gastroenterol. 2015;21(25):7742-53.
- Norman JG, Fink GW, Franz MG. Acute pancreatitis induces intrapancreatic tumor necrosis factor gene expression. Arch Surg. 1995;130(9):966-70.
- Gukovskaya AS, Gukovsky I, Zaninovic V, Song M, Sandoval D, Gukovsky S, et al. Pancreatic acinar cells produce, release, and respond to tumor necrosis factor-alpha. Role in regulating cell death and pancreatitis. J Clin Invest. 1997;100(7):1853-62.
- Habtezion A. Inflammation in acute and chronic pancreatitis. Curr Opin Gastroenterol. 2015;31(5):395-9.
- Kylänpää L, Rakonczay Z Jr, O'Reilly DA. The clinical course of acute pancreatitis and the inflammatory mediators that drive it. Int J Inflam. 2012;2012:360685.
- Klar E, Schratt W, Foitzik T, Buhr H, Herfarth C, Messmer K. Impact of microcirculatory flow pattern changes on the development of acute edematous and necrotizing pancreatitis in rabbit pancreas. Dig Dis Sci. 1994;39(12):2639-44.
- Norman J. The role of cytokines in the pathogenesis of acute pancreatitis. Am J Surg. 1998;175(1):76-83.
- Foitzik T, Hotz HG, Eibl G, Buhr HJ. Experimental models of acute pancreatitis: are they suitable for evaluating therapy? International Journal of Colorectal Disease. 2000;15:127-35.
- Lankisch PG, Pohl U, Otto J, Rahlf G. When should treatment of acute experimental pancreatitis be started? The early phase of bile-induced acute pancreatitis. Res Exp Med (Berl). 1988;188(2):123-9.