D 1195
OULU 2013
U N I V E R S I T Y O F O U L U P. O. B . 7 5 0 0 F I - 9 0 0 1 4 U N I V E R S I T Y O F O U L U F I N L A N D
U N I V E R S I TAT I S
S E R I E S
SCIENTIAE RERUM NATURALIUM
Senior Assistant Jorma Arhippainen
HUMANIORA
University Lecturer Santeri Palviainen
TECHNICA
Professor Hannu Heusala
MEDICA
Professor Olli Vuolteenaho
SCIENTIAE RERUM SOCIALIUM
ACTA
U N I V E R S I T AT I S O U L U E N S I S
Sanna Meriläinen
E D I T O R S
Sanna Meriläinen
A
B
C
D
E
F
G
O U L U E N S I S
ACTA
A C TA
D 1195
EXPERIMENTAL STUDY OF
ACUTE PANCREATITIS
IN A PORCINE MODEL,
ESPECIALLY TIGHT JUNCTION
STRUCTURE AND PORTAL
VEIN CYTOKINES
University Lecturer Hannu Heikkinen
SCRIPTA ACADEMICA
Director Sinikka Eskelinen
OECONOMICA
Professor Jari Juga
EDITOR IN CHIEF
Professor Olli Vuolteenaho
PUBLICATIONS EDITOR
Publications Editor Kirsti Nurkkala
ISBN 978-952-62-0064-4 (Paperback)
ISBN 978-952-62-0065-1 (PDF)
ISSN 0355-3221 (Print)
ISSN 1796-2234 (Online)
UNIVERSITY OF OULU GRADUATE SCHOOL;
UNIVERSITY OF OULU,
FACULTY OF MEDICINE,
INSTITUTE OF CLINICAL MEDICINE, DEPARTMENT OF SURGERY;
INSTITUTE OF DIAGNOSTICS, DEPARTMENT OF PATHOLOGY;
INSTITUTE OF BIOMEDICINE, DEPARTMENT OF ANATOMY AND CELL BIOLOGY;
INSTITUTE OF BIOMEDICINE, DEPARTMENT OF PHYSIOLOGY;
OULU UNIVERSITY HOSPITAL
D
MEDICA
ACTA UNIVERSITATIS OULUENSIS
D Medica 1195
SANNA MERILÄINEN
EXPERIMENTAL STUDY OF ACUTE
PANCREATITIS IN A PORCINE
MODEL, ESPECIALLY TIGHT
JUNCTION STRUCTURE AND
PORTAL VEIN CYTOKINES
Academic dissertation to be presented with the assent
of the Doctoral Training Committee of Health and
Biosciences of the University of Oulu for public defence
in Auditorium 1 of Oulu University Hospital, on 15
February 2013, at 12 noon
U N I VE R S I T Y O F O U L U , O U L U 2 0 1 3
Copyright © 2013
Acta Univ. Oul. D 1195, 2013
Supervised by
Professor Jyrki Mäkelä
Professor Tatu Juvonen
Reviewed by
Professor Matti Eskelinen
Docent Juhani Sand
ISBN 978-952-62-0064-4 (Paperback)
ISBN 978-952-62-0065-1 (PDF)
ISSN 0355-3221 (Printed)
ISSN 1796-2234 (Online)
Cover Design
Raimo Ahonen
JUVENES PRINT
TAMPERE 2013
Meriläinen, Sanna, Experimental study of acute pancreatitis in a porcine model,
especially tight junction structure and portal vein cytokines.
University of Oulu Graduate School; University of Oulu, Faculty of Medicine, Institute of
Clinical Medicine, Department of Surgery; Institute of Diagnostics, Department of Pathology;
Institute of Biomedicine, Department of Anatomy and Cell Biology, Department of Physiology;
Oulu University Hospital, P.O. Box 5000, FI-90014 University of Oulu, Finland
Acta Univ. Oul. D 1195, 2013
Oulu, Finland
Abstract
Acute pancreatitis is a common disease, Finland being among the countries with the highest
incidence. The majority of patients have a mild, self-limiting disease. However, 20% of these
patients develop severe necrotizing pancreatitis with a mortality rate of 7 to 25%. The mechanisms
for developing the severe disease are not known, it is not possible to accurately forecast the
severity of the disease and there is no curative treatment yet.
This study was aimed at analyzing the early phase of acute experimental porcine oedematous
and necrotizing pancreatitis. In Study I, the pancreatic microcirculatory changes were measured
and the expression of tight junction proteins (claudins-2, -3, -4, -5 and -7) and the rate of apoptosis
in the pancreas were all measured. In Study II, bacterial translocation to the blood in the portal
vein blood or to the mesenteric lymph nodes was analyzed and the intestinal expression of tight
junction proteins (claudins-2, -3, -4, -5 and -7) and the intestinal apoptosis/ proliferation rates were
measured. The basic histology of the jejunum and colon were analyzed. Study III analyzed which
cytokines are released from the pancreas to the portal venous blood. In Study IV, the ultrastructure
of the epithelium of the jejunum and colon was analyzed and the expression of adherens junction
proteins, E-cadherin and β-catenin, were measured from both jejunum and colon.
The first study (I) showed that membranous immunoreactivity of claudin-2 in acinar cells
appeared in the pancreas during acute oedematous and necrotizing pancreatitis. The expressions
of claudins -3, - 4, - 5 and 7 were unaffected. The second study (II) showed that bacterial
translocation from the gut was not present at the beginning of acute porcine pancreatitis. The
expressions of claudins-2 and -5 do not become altered; however, there might be some decrease
in claudin-3 expression in the colon and decrease in the expression of claudins-4 and -7 in the
jejunum in necrotizing pancreatitis. Performing the laparotomy itself caused increased apoptosis
in the colon and the jejunum. In the third study (III), the initial inflammatory process was diverse
in oedematous and necrotizing pancreatitis. Increased monocyte count in combination with
elevated PDGF and IL-6 are characteristic of necrotizing pancreatitis in our model. The fourth
study (IV) indicated that necrotizing pancreatitis caused damage to the epithelial and endothelial
cells of the colon in the early stages of the disease. The expression of E-cadherin immunoreactivity
showed a decreasing trend in the colon in both oedematous and necrotizing pancreatitis.
The results of this study suggest that claudin-2 increases in acinar cells during acute porcine
pancreatitis. Bacterial translocation is not present during the early phase of acute porcine
pancreatitis. Increased monocyte count and elevated PDGF and IL-6 are characteristic of early
phase necrotizing porcine pancreatitis and necrotizing porcine pancreatitis causes damage to the
epithelial and endothelial cells of the colon.
Keywords: claudin, cytokine, experimental pancreatitis, porcine, tight junction
Meriläinen, Sanna, Kokeellinen haimatulehdus sikamallissa.
Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta, Kliinisen
lääketieteen laitos, Kirurgia; Diagnostiikan laitos, Patologia; Biolääketieteen laitos, Anatomia ja
solubiologia, Fysiologia; Oulun yliopistollinen sairaala, PL 5000, 90014 Oulun yliopisto
Acta Univ. Oul. D 1195, 2013
Oulu
Tiivistelmä
Akuutti haimatulehdus on yleinen sairaus, jonka ilmaantuvuus Suomessa on verrattain suuri.
Suurimmalla osalla potilaista tauti on lievä ja itsestään paraneva. Kuitenkin 20 %:lle potilaista
kehittyy vaikea haimatulehdus, johon liittyy 7–25 %:n kuolleisuus. On epäselvää, miksi toisinaan kehittyy vaikea tautimuoto. Taudin vaikeusastetta ei voida etukäteen tarkasti ennustaa, eikä
tautiin ole parantavaa hoitoa.
Väitöskirjatyön tarkoituksena oli tutkia lievän ja vaikean haimatulehduksen varhaisvaihetta
kokeellisessa sikamallissa. Työssä I mitattiin haiman mikroverenkierron muutoksia, tutkittiin tiivisliitosproteiinien klaudiini-2:n, -3:n, -4:n, -5:n ja -7:n ilmenemistä sekä apoptoosin määrää
haimassa. Toisessa työssä tutkittiin mahdollista bakteeritranslokaatiota porttilaskimovereen ja
vatsaontelon imusolmukkeisiin, mitattiin suoliston tiivis liitos-proteiinien klaudiinien-2, -3, -4, 5 ja -7 ilmenemistä ja suoliston apoptoosin ja soluproliferaation määrää. Mahdollisia muutoksia
ohut- ja paksusuolen perushistologiassa analysoitiin. Kolmannessa työssä mitattiin sytokiinipitoisuuksia porttilaskimoverestä. Neljännessä työssä analysoitiin ohut- ja paksusuolen mikrorakennetta elektronimikroskopian avulla ja mitattiin vyöliitosproteiinien E-cadherin ja β-catenin
määrää.
I työssä todettiin klaudiini-2:n ilmaantuvan haiman asinaarisolujen solukalvoille lievässä ja
vaikeassa kokeellisessa haimatulehduksessa. Klaudiinien 3,- 4,- 5 ja 7 esiintyminen haimassa ei
muuttunut. II työssä todettiin, että bakteeritranslokaatiota ei tapahtunut seuranta-aikana. Suolistossa klaudiinien-2 ja -5 ilmenemisessä ei tapahtunut muutoksia. Klaudiini-3:n ilmenemisessä
paksusuolessa ja klaudiinien -4 ja -7 ilmenemisessä ohutsuolessa saattaa tapahtua vähenemistä
vaikeassa haimatulehduksessa. Tutkimustoimenpide itsessään aiheutti ohut- ja paksusuolen
apoptoosin lisääntymistä. III työn mukaan tulehdusvaste oli erilainen akuutissa lievässä ja vaikeassa kokeellisessa haimatulehduksessa. Monosyyttimäärän sekä PDGF:n ja IL-6:n pitoisuuksien lisääntyminen, olivat tyypillisiä vaikealle haimatulehdukselle tässä mallissa. IV työssä todettiin, että vaikea haimatulehdus vaurioittaa paksusuolen epiteeli- ja endoteelisoluja. E-cadherin: n
määrässä todettiin jonkin verran vähentymistä sekä lievässä että vaikeassa haimatulehduksessa.
Näiden tulosten mukaan klaudiini-2 lisääntyy sian haiman asinaarisoluissa akuutissa haimatulehduksessa. Sialla ei tapahdu bakteerien translokaatiota haimatulehduksen varhaisvaiheessa.
Sian vaikeaan haimatulehdukseen liittyy monosyyttien, PDGF:n ja IL-6:n lisääntyminen.
Kokeellisessa vaikeassa haimatulehduksessa paksusuolen epiteeli- ja endoteelisolut vaurioituvat
jo varhaisvaiheessa.
Asiasanat: klaudiini, kokeellinen haimatulehdus, sika, sytokiini, tiivis liitos
Acknowledgements
The present study was carried out at the Laboratory Animal Centre, Department
of Surgery, in co-operation with Department of Pathology, Department of
Anatomy and Cell Biology and Department of Physiology, Oulu University
Hospital during the period 2006–2012.
I express my deepest gratitude to my supervisors. I thank Professor Jyrki
Mäkelä for the supportive guidance in every step of this work. I thank Professor
Tatu Juvonen for introducing this subject to me, giving me the opportunity to
engage in this scientific work, his trust and personal help during these years.
I acknowledge the critical appraisal of the manuscript of this thesis on the
part of Professor Matti Eskelinen and Docent Juhani Sand.
I thank Professor Petri Lehenkari for his significant collaboration and
Professors Tuomo Karttunen and Ylermi Soini for their guidance, expertise and
knowledge. I express my gratitude to Professor Karl-Heinz Herzig for his cooperation and the tuition he provided in scientific writing.
I thank Docent Raija Sormunen for her expertise and for her pursuit of
excellence in terms of electron microscopy.
I thank Docent Vesa Koivukangas for his help in this project and for his
excellent teamwork in clinical practice.
I am grateful to my friends and co-workers Riikka Rimpiläinen, MD, PhD
and Merja Vakkala MD, PhD for the essential intellectual and technical cooperation they provided.
I express my appreciation to all my co-authors - Juha Koskenkari MD, PhD,
Docent Vesa Anttila, Professor Juha Risteli, Professor Ilmo Hassinen, Markku
Koskela MD, PhD, Siri Lehtonen MD, PhD, Docent Jussi Rimpiläinen, Toni
Karhu MSc and Meeri Kröger MSc for their expertise and valuable comments. I
thank Pasi Ohtonen MSc for the help with biostatistical analyses and advice.
My sincere thanks go to Hanna Alaoja Jensen MD, PhD, Eija Rimpiläinen
MD, Fredrik Yannopoulos MD, Tuomas Mäkelä MD, Kirsi Alestalo MD and
Sebastian Dahlbacka MD, PhD. You made a vital contribution to this work during
the experimental stages. You were an excellent team and it was a pleasure to be
able to work with you.
I also express my gratitude to R.N. Seija Seljanperä for her great technical
help and mental encouragement. I thank Docent Hanna-Marja Voipio D.V.M,
veterinarian Sakari Laaksonen, technical researcher Veikko Lähteenmäki and the
7
crew of the Laboratory Animal Centre for providing the excellent facilities
associated with this work.
I thank Mrs Erja Tomperi, Mrs Mirja Vahteri and the Biocenter Oulu EM
laboratory staff for their valuable technical assistance during the experimental
laboratory work.
I thank Docent Kari Haukipuro, the Head of the Division of Operative Care
and Heikki Wiik MD, PhD the Chief of Surgery, for the opportunity to do the
scientific work alongside clinical practice.
Docent Juha Saarnio is thanked as the head of the Division of Gastrointestinal
Surgery for the positive attitude towards my scientific work and also for the
mental support during this study. I also wish to thank my brilliant colleagues in
the Division of Gastrointestinal Surgery for their understanding.
I owe my warmest thanks to all my friends. Your companionship has
provided me with the positive energy needed to recharge my batteries and
perspective to correct my horizons. In addition to great friendship, I thank Lotta
Simola MD for her common sense and strong woman power and Hanna
Ronkainen MD, PhD for the intellectual and practical help she provided. I thank
Jussi Ronkainen MSc (Tech.) for the vital technical assistance and excellent
cuisine.
I thank my brother Antti and all the family: Katja, Emilia and Kalle, for
bringing joy and lightness to my life.
My deepest thanks go to my parents Anika and Matti Meriläinen. Your
dedication and hard work in life have set me an example for which I should aim.
Your first priority has always been the well-being of your children and grandchildren, and I am deeply grateful for your unconditional love and support
throughout my life.
This study was financially supported by the Sigrid Juselius Foundation,
Finland, the Finnish Anti-Tuberculosis Association and the Regional Cancer Society
of Northern Finland.
Oulu, December 2012
8
Sanna Meriläinen
Abbreviations
ACS
AJ
APACHE II
ARDS
CAR
CARS
CDE
CECT
CTSI
ERCP
IAH
IAP
ICAM-1
IL
INF-γ
JAM
LBP
MAGI
MODS
MRI
MUPP
NF-κB
NO
OFR
PAF
PRSS1, -2
SAP
SIRS
SPINK1
TER
TFPI
TJ
TLR
TNF-α
WBC
Abdominal compartment syndrome
Adherens junction
Acute Physiology and Chronic Health evaluation II
Acute respiratory distress syndrome
Coxsackie and adenovirus receptor
Compensatory anti-inflammatory response syndrome
Choline deficient ethionine supplemented diet
Contrast enhanced computed tomography
CT-severity index
Endoscopic retrograde cholangiopancreatography
Intra-abdominal hypertension
Intra-abdominal pressure
Inter-Cellular Adhesion Molecule
Interleukin
Interferon- γ
Junctional adhesion molecules
Lipopolysccharide-binding protein
Guanylate kinase with inverted orientation protein
Multiorgan dysfunctions syndrome
Magnetic resonance imaging
Multi-PDZ domain protein
Nuclear factor- κB
Nitric oxide
Oxygen free radical
Platelet activating factor
Protease, serine, -1, -2
Severe acute pancreatitis
Systemic inflammatory response syndrome
Serine protease inhibitor Kazal 1
Transepithelial resistance
Tissue factor pathway inhibitor
Tight junction
Toll-like receptor
Tumor necrosis factor - α
White blood cell
9
VH/CD
ZO
10
Villous height/ crypt depth
Zonula occludens
List of original publications
This thesis is based on the following articles, which are referred to in the text by
their Roman numerals:
I
Meriläinen S, Mäkelä J, Anttila V, Koivukangas V, Kaakinen H, Niemelä E, Ohtonen
P, Risteli J, Karttunen T, Soini Y & Juvonen T (2008) Acute edematous and
necrotizing pancreatitis in a porcine model. Scand J Gastroenterol 43(10): 1259–68
II Meriläinen S, Mäkelä J, Koivukangas V, Jensen HA, Rimpiläinen E, Yannopoulos F,
Mäkelä T, Alestalo K, Vakkala M, Koskenkari J, Ohtonen P, Koskela M, Lehenkari P,
Karttunen T & Juvonen T (2012) Intestinal bacterial translocation and tight junction
structure in acute porcine pancreatitis. Hepatogastroenterology 59(114): 599–606
III Meriläinen S, Mäkelä J, Jensen HA, Dahlbacka S, Lehtonen S, Karhu T, Herzig KH,
Kröger M, Koivukangas V, Koskenkari J, Ohtonen P, Karttunen T, Lehenkari P&
Juvonen T (2012) Portal vein cytokines in the early phase of acute experimental
oedematous and necrotizing porcine pancreatitis. Scand J Gastroenterol. 47(11):
1375–85
IV Meriläinen S, Mäkelä J, Sormunen R, Jensen HA, Rimpiläinen R, Vakkala M,
Rimpiläinen J, Ohtonen P, Koskenkari J, Koivukangas V, Karttunen T, Lehenkari P,
Hassinen I & Juvonen T (2012) Effect of acute pancreatitis on porcine intestine: A
morphological study. Ultrastruct Pathol. In Press
11
12
Contents
Abstract
Tiivistelmä
Acknowledgements
7
Abbreviations
9
List of original publications
11
Contents
13
1 Review of the literature
17
1.1 Anatomy of the human pancreas ............................................................. 17
1.2 Definition and classification of acute pancreatitis .................................. 17
1.3 Epidemiology of acute pancreatitis ......................................................... 18
1.4 Etiology and risk factors of acute pancreatitis ........................................ 19
1.5 Assessment of the severity of acute pancreatitis ..................................... 22
1.6 Pathophysiology ...................................................................................... 24
1.6.1 Pancreatic autodigestion ............................................................... 24
1.6.2 Leukocyte activation and inflammatory mediators ...................... 25
1.6.3 Cytokines and chemokines ........................................................... 26
1.6.4 Toll-like receptors (TLR).............................................................. 28
1.6.5 Nuclear factor- κB (Nf-κB) .......................................................... 29
1.6.6 Plasma cascades involved in inflammation .................................. 30
1.6.7 Acute phase proteins..................................................................... 32
1.6.8 Other mediators ............................................................................ 33
1.6.9 Pancreatic microcirculatory disturbance ...................................... 35
1.6.10 Splanchnic Hypoperfusion ........................................................... 36
1.6.11 Cell death pathways: necrosis and apoptosis ................................ 37
1.6.12 The role of the gut in the pathogenesis of acute
pancreatitis ................................................................................... 38
1.6.13 Abdominal compartment syndrome (ACS) .................................. 49
1.6.14 Systemic inflammatory response syndrome (SIRS) and
Multiorgan dysfunctions syndrome (MODS) ............................... 49
1.7 Clinical course of acute pancreatitis ....................................................... 51
1.8 Experimental models of acute pancreatitis.............................................. 52
2 Aims of the study
57
3 Animals and methods
59
3.1 The Porcine model .................................................................................. 59
3.2 Test animals and preoprative management ............................................. 59
13
3.3
3.4
3.5
3.6
3.7
Anaesthesia, haemodynamic and temperature monitoring ...................... 60
Experimental protocols ........................................................................... 61
Intravital fluorescence microscopy ......................................................... 62
Microdialysis ........................................................................................... 62
Blood samples, portal vein blood cultures and mesenteric lymph
node bacterial cultures............................................................................. 63
3.8 Tissue samples......................................................................................... 63
3.9 Immunohistochemistry and Histopathological analysis .......................... 64
3.10 Apoptosis ................................................................................................ 65
3.11 Cytokine assays ....................................................................................... 66
3.12 Transmission electron microscopy .......................................................... 66
3.13 Statistical analysis ................................................................................... 67
4 Results
69
4.1 Animals excluded from data analysis...................................................... 69
4.2 Comparability of the study groups .......................................................... 69
4.3 Haemodynamic variables ........................................................................ 69
4.4 Blood samples ......................................................................................... 70
4.5 Blood cells............................................................................................... 70
4.6 Portal vein cytokines ............................................................................... 74
4.7 Intravital fluorescence microscopy ......................................................... 78
4.8 Microdialysis ........................................................................................... 79
4.9 Histological severity of the pancreatitis .................................................. 79
4.10 Immunohistochemistry and histology ..................................................... 80
4.11 Apoptosis ................................................................................................ 82
4.12 Cell proliferation ..................................................................................... 82
4.13 Portal vein blood cultures and mesenteric lymph node bacterial
cultures .................................................................................................... 83
4.14 Transmission electron microscopy .......................................................... 83
5 Discussion
87
5.1 The porcine model................................................................................... 87
5.2 Limitations of the study .......................................................................... 88
5.3 Blood cells............................................................................................... 90
5.4 Portal vein cytokines ............................................................................... 91
5.5 Intravital fluorescence microscopy (IVM) .............................................. 94
5.6 Microdialysis ........................................................................................... 94
5.7 Immunohistochemistry and histology ..................................................... 94
5.8 Apoptosis ................................................................................................ 96
14
5.9 Cell proliferation ..................................................................................... 97
5.10 Portal vein blood cultures and mesenteric lymph node bacterial
cultures .................................................................................................... 97
5.11 Transmission electron microscopy .......................................................... 98
6 Conclusions
101
References
103
Original publications
129
15
Fig. 1. The anatomy of the human pancreas (Netter illustration used with permission
of Elsevier, Inc. All rights reserved).
16
1
Review of the literature
1.1
Anatomy of the human pancreas
The pancreas is located between the coeliac trunk and the superior mesenteric
artery. Its head receives blood supply from both of these arteries. The
gastroduodenal artery originates from the coeliac trunk and branches into the
anterior and posterior superior pancreaticoduodenal arteries. The inferior
pancreaticoduodenal artery bifurcates from the superior mesenteric artery and
branches into the anterior and posterior inferior pancreaticoduodenal arteries.
These two vessels anastamose together to form the anterior and posterior arcades.
The dorsal/ superior pancreatic artery has different origins: from the splenic,
coeliac, superior mesenteric or hepatic arteries. It divides into two branches which
anastamose to form the prepancreatic arcade. The right branch runs to the
uncinate process and the left branch forms the inferior/ transverse pancreatic
artery that runs to the cauda of the pancreas. The pancreatic magna artery derives
from the splenic artery and supplies circulation to the cauda (Pansky B 1990).
The pancreatic lobule is supplied by a single end-artery, which usually
supplies first the islets and then the asini (Yaginuma et al. 1986). The pancreatic
capillaries are phenestrated and have increased permeability (Henderson & Moss
1985).
The veins correspond with the arteries with similar arcades. Both sympathic
and parasympathic nerves innervate the pancreas, with all nerves passing through
the coeliac and superior mesenteric plexi. The lymphatic drainage runs in all
directions; the lymphatics follow the artery tree (Pansky B 1990).
The gastrointestinal anatomy of a pig has differences compared to that of
humans. The pig´s pancreas is sizeable. Pigs´ common bile duct and pancreatic
duct enter the duodenum separately while in human they both enter the papilla of
Vater. Pigs´ liver contains six lobes and a gall bladder (Swindle & Smith 1998).
1.2
Definition and classification of acute pancreatitis
The Atlanta Classification system from 1992 was the first universally applicable
classification system for acute pancreatitis (Bradley 1993).
According to the latest revision of the Atlanta Classification, from 2008,
acute pancreatitis diagnosis requires two out of the following three features: 1,
17
abdominal pain suggestive strongly of acute pancreatitis; 2, serum amylase and/
or lipase activity at least three times greater than the upper limit of normal and 3,
characteristic findings of acute pancreatitis on transabdominal ultrasonography or
on contrast enhanced computed tomography (CECT) or magnetic resonance
imaging (MRI). Acute pancreatitis is considered severe during the first week of
the illness if there is persistent systemic inflammatory response syndrome (SIRS)
and/or developing organ failure (Acute Pancreatitis Classification Working
Group, Bollen et al. 2008).
A new international classification of the severity of acute pancreatitis is based
on an international multidisciplinary consultation. In this classification, AP is
divided into four categories of severity: mild, moderate, severe and critical. Local
determinants include (peri)-pancreatic necrosis which can be either sterile or
infected. The diagnosis of necrosis is done with contrast-enhanced computed
tomography. Systemic determinants include organ failure which may be either
persistent or transient (Dellinger et al. 2012).
Fig. 2. Determinant-based classification of the severity of acute pancreatitis (Dellinger
2012, published by permission of Wolters Kluwer Health).
1.3
Epidemiology of acute pancreatitis
Acute pancreatitis is a common disease and its incidence has been rising in the
past two decades in Europe and the USA (Frossard et al. 2008). The annual
18
incidence varies between 5 and 80 people per 100,000. Finland is among the
countries with the highest incidence (Sekimoto et al. 2006, Sand et al. 2009b).
In 80% of cases the disease is mild and self-limiting; 80% of the deaths due
to pancreatitis occur as a result of severe necrotizing pancreatitis (Beger et al.
1997). The overall mortality in acute pancreatitis ranges from 2.1 to 7.8% and the
mortality among necrotizing pancreatitis patients is between 7 and 25%
(Kylanpaa et al. 2010) (Sekimoto et al. 2006). In 12 to 33% of fatal cases the
diagnosis of pancreatitis is made at autopsy (Sekimoto et al. 2006).
Infected pancreatic necrosis develops in 21 to 40% of patients with
necrotizing pancreatitis and the mortality risk associated with infected pancreatic
necrosis varies between 32 and 34% (Petrov et al. 2010, Perez et al. 2002).
Acute severe pancreatitis is associated with a 20 to 80 % prevalence of organ
failure (Kylanpaa et al. 2010). Organ failure is associated with a 32 to 47%
mortality rate. The relative risk of mortality doubles when organ failure and
infected pancreatic necrosis are both present (Petrov et al. 2010, Sekimoto et al.
2006).
1.4
Etiology and risk factors of acute pancreatitis
The two major etiological factors responsible for acute pancreatitis are alcohol
and gall stones. The proportions of pancreatitis attributed to alcohol and gall
stones vary significantly for different countries and regions (Whitcomb 2006a).
The most common cause of acute pancreatitis in Finland is alcohol abuse (70% of
cases) and the second most common is gall stones (20% of cases) (Puolakkainen
et al. 1987). 10% of all cases of pancreatitis are idiopathic and no etiologic factor
can be recognized (Guda et al. 2011).
Gall stone induced pancreatitis is due to transient obstruction of the bile and
pancreatic ducts by gallstone migration. This causes biliary and duodenal fluid
reflux into the pancreatic duct and/ or elevates the hydrostatic pressure in the
pancreatic duct (Frakes 1999). The duration of the duct obstruction contributes to
the severity of the disease (Acosta et al. 1997, Hirano & Manabe 1993, Runzi et
al. 1993, Senninger et al. 1986). Necrosis tends to develop when obstruction
continues for 48 hours or more (Acosta et al. 1997, Acosta et al. 2006, Runzi et
al. 1993b). In the majority of patients (71 to 88%), the stones pass spontaneously
from the main bile duct to the duodenum (Acosta et al. 1997, Acosta et al. 1980).
The mechanisms of alcohol abuse leading to acute pancreatitis are not fully
understood. Alcohol causes a dose-related risk for pancreatitis but, on the other
19
hand, not all heavy drinkers get pancreatitis. The pancreas can metabolize alcohol
to its toxic metabolites (acetaldehyde, fatty acid ethyl esters and reactive oxygen
species) which can induce oxidant stress to the gland. Alcohol and its metabolites
may predispose acinar cells to premature intracellular activation of digestive and
lysosomal enzymes (Apte et al. 2009). Alcohol and its metabolites can destabilize
lysosomes and zymogen granules (Haber et al. 1994), increase digestive and
lysosomal enzyme content (Apte et al. 1995, Grönroos JM et al. 1992), increase
the activation of transcription factors which regulate cytokine expression
(Gukovskaya et al. 2002) and induce a sustained increase in cytoplasmic ionic
calcium causing mitochondrial calcium overload which leads to mitochondrial
depolarization and cell necrosis (Criddle et al. 2006). Some genetic factors may
also influence the risk of alcoholic pancreatitis: PRSS1 and SPINK1 mutations
increase the risk of alcohol induced pancreatitis (Whitcomb 2006b). Alcoholics
with pancreatitis have been reported to possess a polymorphism of a gene that
encodes for carboxyl ester lipase. Mutations of the carboxyl ester lipase gene may
influence fatty acid ethyl ester formation in these patients (Miyasaka et al. 2005).
The protective PRSS2 variant, G191R, is less common among alcoholic
pancreatitis patients compared with controls (Witt et al. 2006). Chronic alcohol
use may predispose to endotoxemia (Bode & Bode 2000), which can trigger
necroinflammation in the pancreas (Apte et al. 2009).
Pancreas divisum may lead to stenosing or inadequately patent minor papilla,
causing incomplete pancreatic drainage and elevated intraductal pressure. Also,
dyscinesia of the sphincter of Oddi may cause high intraductal pressure and
predispose the patient to acute pancreatitis. An intraductal papillary mucinous
tumor might induce acute pancreatitis by mucus or tumor obstruction of the
pancreatic duct (Wang et al. 2009) and ampullary and pancreatic tumors can also
cause acute pancreatitis by duct obstruction. Choledochocele affecting the
pancreatobiliary junction may predispose to acute pancreatitis; the association is
related to stones or sludge obstructing the pancreatic duct. Additionally, other
anomalies can increase the risk of pancreatitis, including an unusually long
common channel between the pancreatic and biliary duct and an annular pancreas
(Guda et al. 2011).
ERCP is associated with a 4 to 8% risk of pancreatitis. Patients with increased
risk of post-erc pancreatitis are characterized as being of female gender with
sphincter of Oddi dyscinesia, normal serum bilirubin, recurrent abdominal pain
and absence of biliary duct dilatation. These factors increase the risk of post-erc
pancreatitis up to 10-fold (Abdel Aziz & Lehman 2007). Young age, recurrent
20
pancreatitis and prior post-erc pancreatitis also increase the risk of post-erc
pancreatitis (Badalov et al. 2009). ERCP procedure-related risk factors include
the number of attemps to cannulate papilla, poor emptying of pancreatic duct after
opacification (Wang et al. 2009) and pancreatic duct injection (Badalov et al.
2009). Pancreatic stent placement is an effective technique to prevent post-erc
pancreatitis (Freeman 2004). The risk of post-erc pancreatitis is higher in low
volume centers but the evidence for physician-associated case load and increased
risk of post-erc pancreatitis is not univocal (Testoni et al. 2011, Loperfido et al.
1998). Trainee participation in the procedure also increases the risk of post-erc
pancreatitis (Cheng et al. 2006).
Additionally, other kind of trauma, including iatrogenic damage, hypothermia
(Mitchell et al. 2003) or ischaemia (Frossard et al. 2008) may induce acute
pancreatitis.
Hypercalcaemia, various drugs, infectious agents (Wang et al. 2009) and
toxins (e.g. scorpion poison) (Frossard et al. 2008) are rare risk factors for acute
pancreatitis. The incidence of drug-induced pancreatitis is low, at 1.4%, and druginduced pancreatitis is seldom necrotizing (14%). Over 100 drugs have been
reported as being associated with an increased risk of pancreatitis (Badalov et al.
2007). Those drugs include: azathioprine, sulfasalazine/ mesalazine, oestrogens,
furosemide, hydrochlorothiatzide and rifampizine (Lankisch et al. 1995). HMGCoA reductase inhibitors (statins) have been reported to cause acute pancreatitis
at any dose and usually after months of therapy. (Singh & Loke 2006) Increased
risk of acute pancreatitis has been associated with viruses (e.g. mumps, coxsackie,
hepatitis B, cytomegalovirus, varicella-zoster and herpes simplex), bacteria (e.g.
mycoplasma, legionella, leptospira and salmonella) fungi (e.g. aspergillus) and
parasites (e.g. toxoplasma, cryptosporidium and ascaris) (Parenti et al. 1996).
Several gene mutations have been found to be associated with an increased
risk of pancreatitis. These include mutations in the genetic coding for cationic
trypsinogen (protease, serine, 1 PRSS1) (Charnley 2003), serine protease inhibitor
Kazal 1 (SPINK1) (Schneider 2005) and cystic fibrosis transmembrane
conductance regulator (CFTR) (Chang et al. 2008). Mutations in genetic coding
for chymotrypsinogen C (CTRC) and calcium-sensing receptor (CASR) increase
the risk of pancreatitis to a lesser extent (LaRusch & Whitcomb 2011). Mutations
in genetic coding for anionic trypsinogen (protease, serine, 2 PRSS2) may have a
protective effect against pancreatitis (Felderbauer et al. 2011). In the development
of acute pancreatitis, early activation of trypsinogen to trypsin is crucial. PRSS1+,
high calcium, low ph and trypsin promote trypsinogen activation. CASR regulates
21
calcium levels. Active trypsin degradation is facilitated by CTRC. SPINK1 blocks
active trypsin and prevents further tissue injury. CFTR promotes the elimination
of trypsin by secretion of fluids which flush it out into the pancreatic duct and
duodenum (Whitcomb 2010).
Autoimmune pancreatitis is a systemic fibro-inflammatory disease which
may appear as acute recurrent pancreatitis. It may also present as biliopancreatic
obstructive disease with or without a pancreatic mass resembling pancreatic
cancer (Frulloni et al. 2011).
Obesity does not predispose to pancreatitis but it is demonstrated that all
major complications are more common and more severe in obese patients. (Evans
et al. 2010) Obesity increases the risk of local complications such as pseudocyst,
abscess and necrosis. It also increases the risk of organ failure and death. The
relative risk is 4.3 for local complications, 2.0 for systemic complications and 2.1
for death (Abu Hilal & Armstrong 2008).
1.5
Assessment of the severity of acute pancreatitis
Predicting the course and severity of the disease in the early stages of acute
pancreatitis remains a challenge. Several scoring systems have been introduced.
The Ranson Score required the measurement of 11 to 22 factors and could only be
calculated after 48 hours surveillance (Ranson et al. 1974, Ranson 1979, Mofidi
et al. 2009). The modified Glascow Criteria and Japanese Severity Score uses
similar variables to the Ranson Score and their accuracy in predicting the severity
and mortality is equal (Imrie 2003, Mofidi et al. 2009). More simple scoring
systems were developed but not validated (e.g. the Clinical Prognostic Staging
System and SAP Score) (Rabeneck et al. 1993, Agarwal & Pitchumoni 1986).
The scoring system developed by Ueda and co-workers is based on three different
variables: serum lactate dehydrogenase, blood urea nitrogen and the findings of a
contrast-enhanced abdominal CT (CECT) performed in the first 2 days after
admission. This scoring system is useful in identifying potential severe acute
pancreatitis patients (SAP) with an extremely severe disease (Ueda et al. 2007).
The Logistic Organ Dysfunction Score (LODS), Marshall Score, Sequential
Organ Failure Assessment Score (SOFA), Acute Physiology and Chronic Health
Evaluation II Score (APACHE II) and APACHE-O all quantify the organ failure
which is associated with acute pancreatitis. They provide help in management
decision making although none of these scores is perfect in predicting the
outcome of a single patient (Mason et al. 2010, Johnson et al. 2004b). APACHE
22
II can be calculated on admission and each day thereafter. A reducing APACHE II
score is an indication of a mild disease whereas a rising score within the first 48
hours is associated with severe pancreatitis (Larvin & McMahon 1989, Wilson et
al. 1990).
The CT Severity Index (CTSI) is based on radiological findings in Contrast
Enhanced Computed Tomography (CECT). CTSI correlates well with the length
of hospital stay, complications and death (Balthazar et al. 1985, Balthazar 2002).
However, CTSI has been criticized since some results failed to show its
correlation with pancreatic necrosis and organ dysfunction (Lankisch et al. 2002),
with renal complications (Mortele et al. 2000) and with clinical outcome (De
Sanctis et al. 1997). The contrast medium itself may increase the risk of renal
failure and even deteriorate the course of the pancreatitis (McMenamin & Gates
1996, Schmidt et al. 1995).
Gadolinium-enhanced MRI seems to be equivalent in assessing the severity
of acute pancreatitis to the use of CECT and the gadolinium is less likely to
aggravate tissue necrosis (Arvanitakis et al. 2004).
Elevated serum amylase and lipase levels are the standard diagnostic tests for
acute pancreatitis but are poor at predicting the severity of the disease (Matull et
al. 2006). There are a few biomarkers that have prognostic value in acute
pancreatitis. C-reactive protein (CRP) is an acute-phase protein that is synthesized
in the liver in response to circulating interleukin-1 and -6 (IL-1, IL-6). At 48
hours after the onset of the symptoms, it is useful in discerning the disease
severity. A cut off level of 150 mg/dl indicates necrotizing pancreatitis with a
sensitivity and specificity of greater than 80% and an accuracy of 86% (Schutte &
Malfertheiner 2008). It is unlikely that acute pancreatitis would be lifethreatening if both the leukocyte count and CRP are normal on hospital admission
(Hämäläinen et al. 2002). However, CRP is not useful in the prediction of organ
failure, infected necrosis or death, nor does it provide very early severity
assessment (Rau 2007, Al Mofleh 2008).
Procalcitonin is also an acute phase protein. Procalcitonin has been proposed
as a marker for predicting the disease severity on admission (Kylanpaa-Back et
al. 2001, Purkayastha et al. 2006). However, the results of its accuracy have been
conflicting (Shafiq et al. 2005).
Trypsinogen activation peptide (TAP) is a small peptide that is cleaved from
trypsinogen during its activation. Urinary TAP 24 hours after admission has
shown a sensitivity of 58 % and specificity of 73 %, in distinguishing between
severe and mild acute pancreatitis (Neoptolemos et al. 2000). Trypsinogen-2 can
23
be measured by a simple urinary dipstick method. It is useful in the diagnosis of
acute pancreatitis but has only 62% sensitivity in the prediction of acute severe
pancreatitis (Lempinen et al. 2001).
Carboxypeptidase B activation peptide (CAPAP) is the largest activation
peptide released from any pancreatic proenzyme. CAPAP could predict the
severity of the disease in serum and urine with 0.52/0.29 sensitivity and 0.73/0.93
specificity (Hjalmarsson et al. 2009).
Interleukin-6 (IL-6) is a cytokine mediating the synthesis of acute phase
proteins (Matull et al. 2006). Interleukin-8 (IL-8) is a secondary mediator of
TNF-α which induces neutrophil activation (Gross et al. 1992). IL-6 has been
shown to detect severe acute pancreatitis with 81to 83.6% sensitivity and 75.6 to
85.3% specificity. IL-8 showed 65.8 to 70.9 % sensitivity and 66.5 to 91.3%
specificity in detecting severe pancreatitis. However, the complexity and high
cost of the assays have limited their clinical use (Aoun et al. 2009).
1.6
Pathophysiology
1.6.1 Pancreatic autodigestion
Under normal conditions, digestive enzymes and enzyme precursors known as
zymogens are packed into zymogen granules; these granules secrete their contents
into the pancreatic duct after eating. In pancreatitis, there is a decrease in
secretion to the duodenum in animals and humans. This is due to decreased apical
secretion of the acinar cells, disruption of the paracellular sealing in the
pancreatic duct allowing the contents to leak into the paracellular space and
redirection of the secretion from the zymogen granules from the apical pole to the
basolateral regions of the acinar cell, as shown by in-vitro and in-vivo animal
studies (Gaisano & Gorelick 2009).
In pancreatitis, intracellular protective mechanisms to prevent trypsinogen
activation or trypsin activity are overwhelmed. The protective mechanisms are the
synthesis of trypsin as the inactive enzyme trypsinogen, the autolysis of activated
trypsin, enzyme compartmentalization, synthesis of specific trypsin inhibitors (eg.
serine protease inhibitor Kazal type 1 SPINK 1) and low intracellular ionized
Ca2+ concentrations as shown with in-vitro studies. Mutations of the SPINK1
gene have been observed in patients with recurrent pancreatitis (Bhatia et al.
2005).
24
Human studies have shown that, after trypsinogen is activated to trypsin,
various enzymes (eg. elastase, phospholipase A2) and the complement and kinin
pathways are activated (Schroder T. 1980, Frossard et al. 2008).
1.6.2 Leukocyte activation and inflammatory mediators
Acute inflammation is a physiological protective response to local injury. The
process of acute inflammation is initiated by cells already present in all tissues,
mainly resident macrophages. These cells activate and release inflammatory
mediators responsible for the clinical signs of inflammation. The acute
inflammatory response requires constant stimulation to be sustained.
Inflammatory mediators have short half-lives and are quickly degraded in the
tissue. Hence, acute inflammation ceases once the stimulus has been removed
(Kumar V et al. 2007).
Localized inflammation is generally tightly controlled by the body at the site
of the injury. Loss of local control or an overly activated response results in an
exaggerated systemic response. This response is called systemic inflammatory
response syndrome (SIRS). The acute phase response is controlled by a decrease
in pro-inflammatory mediators and an increase in endogenous antagonists.
However, when the balance is not accomplished, a massive systemic reaction is
launched. The cytokines becomes deleterious, activating a cascade of reactions
which may lead to multi-organ damage. The prognosis of SIRS or MODS is
challenging to predict since the inflammatory response is very complex and can
be either enhanced or limited by intrinsic processes (Davies & Hagen 1997,
Robertson & Coopersmith 2006)
As shown with rats during the early phase of acute pancreatitis, pancreatic
cells, acinar cells, pancreatic stellate cells, vascular endothelial cells and tissue
macrophages secrete cytokines, chemokines and adhesion molecules that enable
the infiltration of neutrophils, monocytes, macrophages, lymphocytes and
leukocytes into the pancreas. In rats, neutrophils and leukocytes were attracted to
the site of an injury as early as 3 hours after the disease onset (Shanmugam &
Bhatia 2010).
Activation of both innate (granulocytes and especially neuthophils) and
adaptive (mononuclear cells, macrophage, T- and B-lymphocytes) immune
systems is involved in the systemic inflammatory response in acute pancreatitis in
humans. Infiltrating polymorphonuclear neutrophils produce oxygen-free radicals
25
and thus cause local and systemic complications, as shown in rats (Beger et al.
2000).
Monocytes and macrophages play an important role in the pathogenesis of
acute pancreatitis. As shown in mice and rats, the monocytes migrate to the
pancreatic interstitium from the circulation (Shirivastava et al. 2008). Monocytes
and macrophages collected from pancreatitis patients´ blood were shown to
produce cytokines and other inflammatory mediators (Shrivastava & Bhatia
2010). The degree of monocyte activity has also been related to disease severity
since it contributes to the cell death pathways of the pancreatic cells in in-vitro
studies and thus the degree of local damage (Liang et al. 2008).
Systemic inflammation in acute pancreatitis also correlates with the activation
of CD4+ T-helper (Th)-lymphocytes, CD 8+ -lymphocytes and CD 19+ Blymphocytes in rats and humans (Beger et al. 2000, Mora et al. 1997). Th1-cells
produce pro-inflammatory cytokines IL-2, TNF-α and IFN- γ. Th 2-lymphocytes
secrete anti-inflammatory cytokines IL-4, IL-5, IL-6, IL-10 and IL-13 as shown
in-vitro (Lichtman & Abbas 1997).
1.6.3 Cytokines and chemokines
Cytokines are small proteins expressed on the surface of the majority of human
cells and are involved in tissue immunity. In normal conditions, they are found in
low levels in plasma. They function in endo-, para- and autocrine fashion. During
localized inflammation they may leak into the circulation and exceed the amount
of soluble receptors, which may then lead to systemic inflammation (Castellheim
et al. 2009).
In acute pancreatitis, pro-inflammatory cytokines secreted from the
pancreatic cells, vascular endothelial cells and tissue macrophages in the pancreas
enter the circulation through the portal vein and the lymphatic system, as shown
in humans. Cytokines are involved in the development of SIRS. They activate the
vascular endothelium leading to micro-vascular leakage of the capillary veins and
leukocyte migration into the tissues, as seen in experimental rat pancreatitis. Proinflammatory cytokines also activate the coagulation process in human patients
which causes thrombosis in the small vessels of many organs. Both of these
phenomena result in impaired tissue microcirculation and may lead to organ
failure in severe pancreatitis (Kylanpaa et al. 2010).
In humans, a dynamic balance exists between the pro- and anti-inflammatory
cytokines. Anti-inflammatory cytokines mediate the compensatory anti26
inflammatory response (CARS), which also activates early, and this may control
the systemic inflammatory reaction. Immunosuppression is also partially due to
monocyte anergy, in which monocytes produce lesser amounts of proinflammatory cytokines. Excessive CARS leads to immune-suppression and
predisposition to infectious complication in the later phase of the disease
(Mentula et al. 2004, Beger et al. 1986, Kylanpaa et al. 2010).
Several cytokines have been found to be associated with the pathogenesis of
pancreatitis in humans during hospital admission and the later phase of the
disease. These include the following pro-inflammatory cytokines: tumour
necrosis factor-α (TNF-α), interferon- γ (IFN- γ), interleukins- 1β, -2, -6 and -8
(IL-1β, L-2, IL-6 and IL-8). IL-10 is the most potent anti-inflammatory cytokine.
IL-1ra and IL-6 are also important anti-inflammatory cytokines and IFN- γ and
IL-4 can act as anti-inflammatory cytokines as well (Kylanpaa et al. 2010,
Kilciler et al. 2008, Shen et al. 2011, Hayashi et al. 2007).
There are a few previous works concerning the presence of cytokines in
porcine pancreatitis 6 to 72 hours following the onset of the disease. TNF-α, IL-1,
IL-1β, IL-6 and IL-10 have all been shown to rise after 6 to 24 hours of severe
acute pancretitis in pigs (Yekebas et al. 2002, Yang et al. 2004, Yan et al. 2006, Li
et al. 2003, Li et al. 2007, Tao et al. 2008). The cytokines were measured from
peripheral venous blood in all but one study. Li et al. measured IL-1 and IL-6
from serum drawn from vena cava and portal vein (Li et al. 2003).
TNF-α is generated in blood monocytes and macrophages. It mediates the
release of various inflammatory mediators and has a strong toxic effect. It induces
the effect of Inter-Cellular Adhesion Molecule 1 (ICAM-1) expressed in the
membranes of endothelial cells and leukocytes. Activated leukocytes bind to the
endothelial cells with ICAM-1 when transmigrating through the endothelium.
TNF-α also downregulates the expression of thrombomodulin in endothelial cells,
activates the coagulation system, increases thrombosis and impairs the endothelial
cells causing increased capillary permeability (Zhang et al. 2009).
IFN- γ may have an anti-inflammatory effect on acute pancreatitis. IFN- γ (-/) mice exhibited exacerbated cerulein-induced pancreatic injury with enhanced
neutrophil recruitment. IFN- γ (-/-) mice also presented exaggerated and
prolonged NF-κB activation and increased pancreatic gene expression of TNF-α
and COX-2. When administered 4 hours after the cerulean injection, IFN- γ
attenuated acute pancreatitis in both wild type and IFN- γ (-/-) mice (Hayashi et
al. 2007). Treatment with IFN- γ was found to attenuate the progression of
27
intrapancreatic necrosis and reduce neutrophil tissue infiltration within the first 24
hours after the induction of severe acute pancreatitis in rats (Rau et al. 2006).
Interleukin-6 and -8 (IL-6 and IL-8) are the most studied cytokine and
chemokine in acute pancreatitis. They have been shown to possess an early
prognostic value for the severity of the disease. At the moment, they are in limited
clinical use (Aoun et al. 2009). IL-6 has both pro- and anti-inflammatory effects.
It stimulates the synthesis of acute phase proteins in the liver (Castell et al. 1989)
and, on the other hand, it prevents the synthesis of pro-inflammatory cytokines
IL-1β and TNF-α (Opal & DePalo 2000). IL-8 is synthesized by monocytes,
endothelial cells and neutrophils. It is a pro-inflammatory chemokine that plays a
role in neutrophil chemoattraction, degranulation and the release of elastase
(Gross et al. 1992, Aoun et al. 2009).
IL-1 is mainly secreted by macrophages and is a pro-inflammatory cytokine
that can activate neutrophils and the endothelium. It promotes fever, hypotension,
increased capillary permeability and the release of other cytokines (Wilson et al.
1998).
IL-2 is produced by T-lymphocytes and causes monocyte activation, T-helper
cell recruitment and monocyte- T-cell interaction. IL-2 concentration is depressed
in many pathological conditions, such as burns, experimental sepsis and
experimental pancreatitis (Wilson et al. 1998).
Interleukin-10 (IL-10) is a cytokine that inhibits the synthesis of Th1 cells. It
inhibits the release of pro-inflammatory cytokines and inhibits the activity of
monocytes and macrophages (Zhang et al. 2007).
1.6.4 Toll-like receptors (TLR)
Toll-like receptors (TLRs) are trans-membrane receptors for surface signal
transduction associated with the body´s inflammatory response (Zhang et al.
2010).
During the early stages of acute pancreatitis, the expression of TLR4 in rats´
pancreatic duct epithelium and pancreatic vascular endothelium increases (Li et
al. 2005). In-vitro human studies have shown that inhibiting TLR4 inhibits the
increase of TNF-α concentration, suggesting that TNF-α release is mediated by
TLR4 (Hietaranta et al. 2005). This suggests that TLR4 has a role in the
activation of the immune system during acute pancreatitis. TLR4- and TLR9deficient mice exhibited acute pancreatitis with reduced pancreatic tissue damage
compared to wild type and TLR2-deficient mice, suggesting a role for TLR4 and
28
TLR9 but not TLR2 in the pathogenesis of acute pancreatitis (Awla et al. 2011,
Hoque et al. 2011). Pre-treatment with TLR9 antagonist reduced pancreatic
oedema, inflammatory infiltrate and apoptosis in wild type mice (Hoque et al.
2011).
It has been hypothesized that possible trigger factors for TLR4 up-regulation
in acute pancreatitis might include enzymes released from pancreatic cells,
bacteria and toxins from the intestine after gut barrier decline (pathogenassociated molecular patterns a.k.a. PAMPs, e.g. endotoxin) and cytokines and
inflammatory mediators released in an inflammatory reaction (Zhang et al. 2010).
TLR4 seems to affect the progression of acute pancreatitis. The rapid
degradation of heparin sulphate in the extracellular matrix with pancreatic
elastase is able to activate the TLR4 signalling pathway, eventually causing SIRSlike symptoms in mice (Johnson & Abu-Hilal 2004). It was shown in–vitro that
the TLR4 pathway activates NF-κB and subsequently leads to the activation of
corresponding immunocytes to release pro-inflammatory cytokines (Yamamoto et
al. 2002).
The expression of TLR4 was shown to be up-regulated in the kidneys, liver
and lungs in acute experimental pancreatitis in rats and mice, suggesting that it
plays a part in the pathogenesis of organ failure (Zhang et al. 2010). TLR2 and
TLR4 proteins were increased in the ileal mucosa of rats with acute pancreatitis
during the first 6 hours and decreased after 12 hours, correlating with the levels of
NF-κB suggesting TLR2/4´s role in the intestinal immune response (Sawa et al.
2008).
TLR2 recognizes the lipoproteic acid (LTA) of gram-positive bacteria. The
expression of TLR2 and TLR4 were decreased in macrophages from
bronchoalveolar lavage 6 hours after the onset of the disease in rats. TNF-α
release from these macrophages was also diminished. This suppression of the
immune response in the early phase of acute pancreatitis may correlate with the
mechanisms of infectious complications (Matsumura et al. 2007).
1.6.5 Nuclear factor- κB (Nf-κB)
Nf-κB is a multi-functional transcription factor that plays an important role in the
initiation of innate and adaptive immune responses. Nf-κB is activated by
numerous stimuli and contributes to the transcription of numerous genes involved
in inflammation, apoptosis, cell proliferation and differentiation. Nf-κB is
involved in the activation of many pro-inflammatory genes, such as the genetic
29
coding for cytokines (e.g. TNF-α, IL-1β and IL-2) chemokines (e.g. IL-8),
adhesion molecules (e.g. ICAM-1) and acute phase proteins (Castellheim et al.
2009). Stimulated Nf-κB binds to promoters or enhancers of the target genes in
the nucleus, resulting in increased transcription and expression (Liu & Malik
2006).
Nf-κB is activated early in the pancreas in experimental pancreatitis with
mice and rats. The activation is biphasic, the first phase being 30 minutes after the
onset of the pancreatitis. After 90 min, the Nf-κB level returned to near basal
levels. The second phase of activation occurred between 3 and 6 hours after the
onset (Rakonczay et al. 2008).
Animal models with rats and mice have identified factors such as proinflammatory cytokines, cholecystokinin (CCK) or reactive oxygen species
(ROS) that can stimulate Nf-κB in the pancreas in acute pancreatitis. Nf-κB
activation has also been shown in the liver, lungs, infiltrating mononuclear cells
and endothelium in acute experimental pancreatitis (Rakonczay et al. 2008).
Animal models with rats have shown that inhibiting Nf-κB during acute
pancreatitis can inhibit the expression and release of inflammatory factors,
increase apoptosis of the pancreatic cells, reduce necrosis, decrease the severity of
the disease and reduce mortality (Dunn et al. 1997, Gukovsky et al. 1998).
There are only a few studies concerning Nf-κB activation in acute
pancreatitis. The mononuclear cells in peripheral blood from acute pancreatitis
patients showed increased levels of Nf-κB activation. Nf-κB activation was
similar in both mild and severe diseases (Satoh et al. 2003, O'Reilly et al. 2006).
1.6.6 Plasma cascades involved in inflammation
Complement system
The complement system´s antibacterial properties are due to opsonization,
phagosytosis and the subsequent killing of the pathogen, in some cases causing
lysis or the promotion and coordination of inflammatory events (chemotaxis,
leukocyte activation). Complement can be activated by classical, lectin or
alternative pathways. C5a is a potent and multifunctional split product generated
by the activation of all three pathways (Castellheim et al. 2009).
30
Complement activation occurs early in human pancreatitis. The complement
inhibitor CD59 was already elevated at hospital admission and the degree
correlated with the disease severity (Lindström et al. 2008).
Interruption of C5a action, either by deletion of its receptor or the protein,
resulted in the worsening of acute pancreatitis in mice. The lung injury associated
with pancreatitis also got more severe when C5a action was inhibited (Bhatia
2009).
Coagulation system
Coagulation can be activated by two pathways: the factor XII-dependent intrinsic
pathway and the tissue factor dependent extrinsic pathway. The end product,
fibrin, is involved in clot formation. Pro-inflammatory cytokines can induce tissue
factor expression on endothelial and mononuclear cells. The coagulation product,
thrombin, further activates the production of pro-inflammatory cytokines by NFκB (Castellheim et al. 2009). Plasma tissue factor (TF) and tissue factor inhibitor
(TFPI) levels were elevated in patients with acute pancreatitis on hospital
admission, correlating with the severity on the disease. TF/TFPI ratios were lower
in non-survivors than in survivors (Sawa et al. 2006, Yasuda et al. 2009). Free
TFPI levels and free/ total TFPI ratios were higher in non-survivors. Failure of
TF-initiated thrombin generation, explained by high levels of free TFPI, may be
associated with OF and mortality in acute pancreatitis (Lindstrom et al. 2011).
Fibrinolytic system
The fibrinolytic system resolves a clot with plasmin degrading fibrin. Three
anticoagulant pathways prevent the systemic activation of coagulation in normal
physiological circumstances: antithrombin, activated protein C (APC) and TFPI.
During inflammation, all these pathways can be impaired leading to disseminated
intravascular coagulation (DIC), which is essential in the pathogenesis of MODS
(Levi & van der Poll 2005).
The impairment in the APC pathway during the first 2 weeks of hospital care
has been shown to be associated with the development of organ failure in patients
with severe acute pancreatitis (Lindstrom et al. 2006). APC treatment improved
the survival rate in severe acute pancreatitis in a rat model but failed to show
benefits in human patients (Alsfasser et al. 2006, Pettila et al. 2010).
31
Kallikrein-kinin system
The kallikrein-kinin system produces bradykinin on the endothelial cell surface.
Bradykinin has a dual effect on microvessels by stimulating nitric oxide (NO)
production. In low-concentration, it induces vasodilation whereas high
concentrations cause vasoconstriction. The kallikrein-kinin system is also linked
to the coagulation and complement systems (Castellheim et al. 2009).
In rats, treatment with a bradykinin B2 receptor antagonist increased the
number of pancreatic capillaries, stabilized capillary blood flow, decreased the
average number of adherent leukocytes in micro-veins and improved pancreatic
microcirculation, suggesting that the suppression of bradykinin formation
improves microcirculatory disturbance (Blöehle et al. 1998). On the other hand,
the inhibition of bradykinin increased the severity of oedematous pancreatitis in
rats (Weidenbach et al. 1995). As shown with rat studies, the treatment with a
bradykinin B2 receptor antagonist is effective in preventing microcirculatory
events, only if started before oedema formation (Griesbacher et al. 2000).
The kallikrein-kinin system is activated in human plasma and peritoneal fluid
in pancreatitis and the activation is more pronounced in the severe disease form
(Lasson & Ohlsson 1984). Six hours after the onset of porcine pancreatitis, the
kallikrein-kinin system was activated in the pig´s peritoneal fluid but not in its
plasma (Ruud et al. 1985).
1.6.7 Acute phase proteins
During inflammation in humans, pro-inflammatory cytokines stimulate liver cells
to synthesize multiple acute phase proteins, such as C-reactive protein (CRP),
fibrinogen, pro-calcitonin, LPS-binding protein (LBP) and complement and
coagulation proteins. While the production of these acute phase proteins in the
liver is increased, the production of other proteins like albumin, transferrin,
cholesterol and fatty acids is decreased (Castellheim et al. 2009, Khan et al.
2012).
32
1.6.8 Other mediators
Nitric oxide (NO)
NO is a small inorganic compound and a free radical. NO synthases cNOS, eNOS
and iNOS catalyze the formation of NO from L-arginine (Chvanov et al. 2005).
Low doses of NO, catalyzed by cNOS, improve whereas high doses of NO,
catalyzed by iNOS, aggravate the microcirculatory disturbance in different
organs. High levels of NO are generated after acute necrotizing pancreatitis in
rats, aggravating pancreatic tissue injury (Zhang et al. 2007a). Small doses of NO
are protective, improving microcirculation in acute pancreatitis in mice and rats
(DiMagno et al. 2004, Dobosz et al. 2005).
The level of NO was significantly higher in the plasma of patients with severe
acute pancreatitis 24 to 48 hours after hospital admission than in mild pancreatitis
or control patients (Que et al. 2010, Zeng XH et al. 2002).
Intercellular adhesion molecule-1(ICAM-1)
ICAM-1 is an adhesion molecule expressed in vascular endothelial cells. In
normal circumstances, it is absent or little expressed. During inflammation, its
expression is upregulated and it induces leukocyte-endothelial cell adhesion,
facilitating the leukocyte migration. It also causes increased capillary
permeability, reduced capillary blood flow and pancreatic microcirculatory
disorder, as shown in experimental pancreatitis with rats (Zhang et al. 2009a).
Blocking ICAM-1 has been shown to protect against local and systemic organ
damage in experimental acute pancreatitis in rats (Bhatia 2009).
In human patients, ICAM-1 peak concentration values were higher and the
peak values occurred later in severe pancreatitis patients than in mild pancreatitis
(6 days rather than 1 day after hospital admission) (Kaufmann et al. 1999).
Oxygen free radicals (OFRs)
Only a few OFRs are produced under normal conditions. The activation of
polymorphonuclear neutrophils induces the generation of large amounts of
oxygen free radicals. Oxygen free radicals harm the capillary endothelial cells,
increase the capillary permeability, enhance the ICAM-1 mediated leukocyte33
endothelial cell interaction and cause pancreatic microcirculatory disturbance in
experimental pancreatitis in rats (Sunamura et al. 1998, Zhang et al. 2009a).
Increased levels of markers for oxidative stress and decreased antioxidant
levels were observed 24 hours after hospital admission in patients with
pancreatitis. The degree of change correlated with the severity of the disease
(Abu-Zidan et al. 2000, Dziurkowska-Marek et al. 2004).
Platelet activating factor (PAF)
An inactive form of PAF is located on the cell membrane in pancreatic or
systemic endothelial cells, macrophages or platelets. TNF and OFRs promote the
secretion of PAF. PAF acts via specific cell surface receptors, which have been
identified in many tissues and cells such as platelets, leukocytes and endothelial
cells. PAF is a potent platelet aggregation-stimulating factor (Zhang et al. 2009a).
PAF plays a role in the microcirculatory disorder in acute pancreatitis
(Flickinger & Olson 1999). It can reduce blood flow and increase both
histological severity and pancreatic leukocyte recruitment in rabbits and rats
(Yotsumoto et al. 1994, Zhou et al. 2002).
PAF receptor antagonists have shown promise in animal studies by blocking
PAF-mediated inflammatory injury. Lexipafant, a PAF antagonist, showed
promise also in phase II human trials but failed to show improvement in the phase
III trial (Chen et al. 2008).
Endotoxin
Endotoxin is a constituent of the outermost layer of enteric gram-negative
bacteria. It is released in bacterial lysis and it is a potent stimulator for the release
of pro-inflammatory cytokines from leukocytes and endothelial cells (De Beaux
& Fearon et al. 1996).
Endotoxin is normally restricted to the lumen of the intestine by the gut
barrier. When entering the circulation, endotoxin binds to circulating
lipopolysaccharide-binding protein (LBP) or CD14 and activates monocytes and
macrophages. Toll-like receptor-4 is a co-receptor for CD14 leading to endotoxin
mediated NF-κB activation and the synthesis and release of pro-inflammatory
mediators such as IL-1, IL-6, IL-8 and TNF-α (Schletter et al. 1995, Chow et al.
1999).
34
Endotoxaemia is known to occur in a proportion of acute pancreatitis
patients. This may be due to the episodic release of endotoxin and also its rapid
clearance from the circulation (De Beaux & Fearon 1996).
In acute pancreatitis, endotoxaemia is associated with poor prognosis.
Patients with high endotoxin concentrations followed a more severe disease
course (Exley et al. 1992) and patients who developed multiple organ failure had
decreased anti-endotoxin antibodies with increased endotoxin concentrations
(Windsor et al. 1993).
At the time of admission, patients with severe acute pancreatitis were found
to have significantly depressed anti-enterobacterial common antigen antibody
titres compared to both patients with mild disease and controls. There was a
significant rise in the titre during the course of the disease in those patients who
survived whereas the rise was absent in those who died (Kivilaakso et al. 1984).
Enteral feeding of acute pancreatitis patients prevented the increase of serum
IgM anti-endotoxin antibodies, which was seen in parenterally-fed patients.
Enteral feeding reduced the need for surgery and the risk of intra-abdominal
sepsis, MODS and mortality (Windsor et al. 1998).
1.6.9 Pancreatic microcirculatory disturbance
The pancreas is supplied by a rich plexus of arteries derived from the coeliac and
superior mesenteric arteries. The pancreatic lobule is supplied by a single endartery, which usually supplies first the islets and then the asini (Yaginuma et al.
1986). The pancreatic capillaries are phenestrated and have increased
permeability (Henderson & Moss 1985). Pancreatic tissue is very sensitive to
ischaemia and one etiology of acute pancreatitis is ischaemia. Also, complete
venous occlusion may lead to acute pancreatitis (Waldner 1992).
In porcine mild experimental pancreatitis, the pancreatic tissue oxygen
tension increased 1 to 6 hours after the induction of the disease (Kinnala et al.
2001, Kinnala et al. 2002). Mild experimental pancreatitis in rats was associated
with relative hyperaemia (Foitzik et al. 1994a). Severe experimental porcine
pancreatitis, on the other hand, is associated with reduced capillary perfusion
measured by microangiography (Nuutinen et al. 1986). Severe experimental rat
pancreatitis was associated with reduced pancreatic oxygen tension and reduced
oxygen saturation (Liu et al. 1996, Bassi et al. 1994, Foitzik et al. 1994a). In dogs
with severe acute pancreatitis, pancreatic oxygen consumption reduced,
suggesting decreased pancreatic circulation (Donaldson & Schenk 1979).
35
Vasoconstriction is an early event in acute pancreatitis. The first signs of the
microcirculatory vasoconstriction were already visible 2 minutes after the onset of
the disease in rats. Leukocyte adherence, forming plaques on the walls of the
interlobular veins, was seen after 6 minutes. Vasoconstriction is subsequently
followed by vasodilatation (Kusterer et al. 1993). However, other studies have
reported decreased pancreatic blood flow enduring the whole surveillance time of
2 to 8 hours (Plusczyk et al. 2003, Cuthbertson & Christophi 2006). Re-perfusion
injury may play an important role in the acinar cell damage (Vollmar & Menger
2003). Increased levels of markers for oxidative stress and decreased antioxidant
levels were observed 24 hours after hospital admission in patients with
pancreatitis. The degree of change correlated with the disease severity (AbuZidan et al. 2000, Dziurkowska-Marek et al. 2004).
Microvascular derangements in humans have been studied indirectly.
Histological analyses from surgically resected pancreatic necrosis have revealed
that vessel wall media necrosis is present and that polymorphonuclear leucocytes
have infiltrated the vessel wall. Intravascular microthrombi exist and
extravasation of contrast medium has been seen in microangiography
(Cuthbertson & Christophi 2006).
Functional capillary density is reduced but non-nutritive perfusion through
shunts is maintained in dogs. Fenestration is increased in rats. Thoracic duct flow
is first increased but then slows to abnormal levels. Blood cells and debris may
block the lymph flow (Cuthbertson & Christophi 2006).
In humans, pancreatic necrosis develops 24 to 48 hours after the onset of
symptoms. However, necrosis becomes more easily recognized in CT scans 2 to 3
days after onset (Balthazar 2002, Nuutinen et al. 1988).
1.6.10 Splanchnic Hypoperfusion
Microcirculatory disorder affects other organs as well as the pancreas; it can be
found in the colon, liver and lungs from the early phase of the disease, persisting
for 48 hours and more in rats. Microcirculatory disorder includes reduced
capillary blood flow, increased permeability and increased leukocyte endothelial
interaction (Foitzik et al. 2002). Increased capillary permeability causes
haemoconcentration and reduced circulating volume in rats (Foitzik et al. 2000).
Haemoconcentaration was shown to precede central haemodynamic alterations in
porcine pancreatitis during 6 hours surveillance (Kinnala et al. 1999).
Tachycardia, hypotension, decreased cardiac output and decreased central venous
36
pressure are common findings in acute experimental pancreatitis (Cuthbertson &
Christophi 2006, Yegneswaran et al. 2011).
Splanchnic hypoperfusion is present early in acute porcine haemorrhagic
pancreatitis. The Pco2 gap between intestinal tonometric and arterial samples
increased after 180 minutes from the onset of pancreatitis (Juvonen et al. 1999).
Reduction of intestinal blood flow and colonic mucosal ischemia are seen after 6
hours in acute experimental pancreatitis in rats (Wang et al. 1996). Splanchinic
hypoperfusion seems to correlate with disease severity, since reduced
microcirculatory blood flow in the colon has been seen in severe and maintained
capillary blood flow in mild experimental acute pancreatitis in rats (Foitzik et al.
2002, Hotz et al. 1998). Also, a lowered gastric intramucosal pH, suggesting
gastric mucosal ischaemia, has been shown in acute pancreatitis patients 24 to 48
hours after hospital admission (Juvonen et al. 2000b).
Decreased renal blood flow leads to renal failure and tubular necrosis, and
reduced splanchnic blood flow causes intestinal ischaemia and hepatic
hyperaemia. Increased coagulation predisposes the patient to, for example, splenic
vein thrombosis (Cuthbertson & Christophi 2006).
1.6.11 Cell death pathways: necrosis and apoptosis
Cell death is defined by the irreversible loss of membrane integrity (Kroemer et
al. 2005). Two modes of cell death, apoptosis and necrosis, occur in pancreatic
acinar cells in acute pancreatitis. The balance between apoptosis and necrosis
might be related to the severity of the disease. In oedematous pancreatitis,
apoptosis is the main form of cell death whereas necrosis predominates in severe
pancreatitis (Bhatia 2004a). Inducing acinar cell apoptosis in mice with acute
pancreatitis ameliorated the severity of the disease (Bhatia et al. 1998).
Apoptosis is a genetically regulated, programmed cell death which does not
lead to release of the cellular contents and does not evoke inflammation. In
apoptosis, the cell and organelles shrink, nuclear chromatin condenses and DNA
is cleaved. Necrosis leads to leakage of the cellular contents and thus provokes
inflammation in the surrounding tissue (Criddle et al. 2007).
Apoptosis occurs mainly by extrinsic (receptor-mediated) or intrinsic
(mitochondrial) pathways. The extrinsic pathway is activated by the ligation of
transmembrane death receptors (e.g. TNF receptor) which then causes activation
of activator and effector caspases. The intrinsic pathway is initiated by the
37
disruption of the mitochondrial membrane and the release of mitochondrial
proteins (Bhatia 2004b).
Apoptosis may be converted to necrosis in acinar cells by neutrophils, as
shown in rats (Sandoval et al. 1996).
A cytosolic Ca2+ rise is involved in both apoptosis and necrosis. Intracellular
2+
Ca controls normal secretions in pancreatic acinar cells. Ca2+ is mainly stored in
the endoplasmic retinaculum (ER). Abnormal release of Ca2+ from the ER to
cytosol leads to the intracellular activation of trypsinogen and other digestive
enzymes. It also leads to NF-κB activation and proinflammatory cytokine release
from the acinar cells (Raraty et al. 2005). Oscillatory Ca2+ rises may induce
apoptosis (Gerasimenko et al. 2002) and persistent cytosolic elevation induces
necrosis (Criddle et al. 2006).
Beside Ca2+ accumulation, Na+ overloading and changes in the mitochondrial
permeability are also involved in necrosis (Levitsky et al. 1998, Lemasters 1999,
Padanilam 2003).
In acute experimental rat pancreatitis, apoptosis occurs in other organs as
well, such as the liver, small intestine, kidney, lung and thymus, 5 to 6 hours after
the disease onset. The degree of apoptosis correlates with the severity of organ
damage (Hori et al. 2000, Zhang et al. 2007b, Zhang et al. 2008, Takeyama 2005,
Takeyama et al. 1998).
The resolution of multiorgan dysfunction is dependent on neutrophil
apoptosis. Delayed neutrophil apoptosis is associated with ongoing inflammation
and further tissue damage. In human patients, delayed neutrophil apoptosis was
associated with mild and, above all, with severe acute pancreatitis (O'Neill et al.
2000).
1.6.12 The role of the gut in the pathogenesis of acute pancreatitis
Increased gut permeability is shown to correlate with disease severity both in a rat
model (Ryan et al. 1993) and with human patients (Liu et al. 2008). With
pancreatitis patients, gut permeability to macro- and micromolecules has been
shown to be increased 24 hours after the onset of symptoms (Liu et al. 2008,
Juvonen et al. 2000a). Systemic endotoxin levels have been associated with the
degree of permeability disorder and with the severity of the disease in humans
(Ammori et al. 1999, Ammori et al. 1999, Exley et al. 1992, Windsor et al. 1993).
Gastric intramucosal pH levels also correlate with levels of antibodies to
endotoxin in human patients, suggesting that permeability disorder is related to
38
mucosal ischaemia (Soong et al. 1999). There are no studies on simultaneous
changes of gut mucosal circulation and permeability.
In experimental animal models, gut permeability is shown to rise as early as 3
to 6 hours after the onset of acute pancreatitis (Leveau et al. 2005) (Andersson et
al. 1998, Wang et al. 1996). With sensitive measures like intravital microscopy
and fluorimeter measurement, it has been shown that bacterial translocation
occurs between 3 and 6 hours after the induction of pancreatitis in rats (Samel et
al. 2002, Lutgendorff et al. 2009). Enteric bacteria have been cultured from rats’
mesenteric lymph nodes, liver, spleen and pancreas 24 to 48 hours after the onset
of the pancreatitis (Runkel et al. 1991, Tarpila et al. 1993, Cicalese et al. 2001).
In the human disease, pancreatic necrosis is often infected with gramnegative enteric bacteria suggesting bacterial translocation from the gut (Beger et
al. 1986) but the exact route of the bacterial invasion remains obscure. The route
could be lymphatic, transperitoneal or haematogenous. In humans, correlation has
been found between intestinal barrier dysfunction, the development of
bacteraemia and mortality from acute pancreatitis (Besselink et al. 2009a).
Median detection of bacteraemia was day seven after hospital admission
(Besselink et al. 2009b). Microbial contamination of the pancreatic necrosis is
more common in biliary than alcohol-induced pancreatitis (Räty et al. 1998).
Other evidence also exists: bacterial DNA was not observed with PCR in
blood drawn from human pancreatitis patients at hospital admission and 3 and 7
days after (Ammori et al. 2003a).
The gut may promote and aggravate pancreatitis-related multi-organ failure in
nonbacterial mechanisms. Studies of ischaemia-reperfusion with rats and pigs
have shown that the gut may enhance the priming and over-activation of
circulating leukocytes and gut-associated macrophages which then release
cytokines and aggravate the MODS (Koike et al. 1995, Sarin et al. 2004).
It has been shown that the intestinal lymphatics mediate nonbacterial, tissue
injurious gut-derived factors, which can induce acute respiratory distress
syndrome (ARDS) and MODS after conditions associated with significant
splanchnic ischaemia in rats, pigs and baboons. The factors in the lymph causing
these harmful systemic effects through iNOS and TLR4-dependent pathways are
yet to be fully identified (Deitch 2010, Zhou et al. 2010).
39
Intestinal barrier
The intestinal barrier has several tasks: it must allow active absorption of
nutrients and allow passive paracellular transportation based on concentration
gradients. On the other hand, it must also allow the secretion of waste products
and inhibit the transmigration of luminal microbes (Shen et al. 2009).
The epithelial barrier is controlled by transcellular and paracellular pathways.
The energy-dependent transporters and channels in apical and basolateral cell
membranes control the transcellular pathway. Amino-acids, electrolytes, shortchain fatty acids and sugars are actively transported by the trans-cellular pathway.
The paracellular pathway is controlled by the intercellular complexes which
regulate the passive diffusion of ions and small solutes through the paracellular
space (Groschwitz & Hogan 2009b).
Glucose is actively transported with Na+ across the apical membrane by the
Na+K+-ATPase pump and then to the paracellular space by glucose transport. The
accumulation of these osmotically active solutes in the paracellular space enables
the absorption of water via the paracellular pathway. Glucose uptake continues to
increase even after saturation of the transcellular pathway; the increase occurs due
to solvent-drag across the paracellular pathway (Madara 1990).
The epithelial barrier function can be measured by trans-epithelial electrical
resistance (TER) and by orally administered probes that are then measured from
the urine. TER is the most sensitive measure of mucosal barrier function; it
reflects the degree to which ions traverse tissue. Therefore, TER largely reflects
the paracellular resistance (Blikslager et al. 2007).
The gut paracellular permeability can be quantified by orally given probes
that are then measured from the urine. These probes can selectively reflect
permeability from different sites in the gut. Certain saccharides (sucrose,
lactulose, rhamnose, cellobiose and mannitol) are destroyed in different parts of
the gut, allowing regional analysis of permeability (Teshima & Meddings 2008).
A variety of mechanisms contribute to the gut barrier. These are non-specific
luminal mechanisms, intestinal epithelial cells and the immunologic response.
Gastric acid and digestive enzymes inhibit bacterial overgrowth. Intestinal
motility and mucus, secreted by intestinal epithelial cells, diminish bacterial
adherence to the epithelial cells. IgA binds foreign antigens in the gut lumen and
defensins are directly toxic anti-microbial peptides which disrupt the cell
membranes of bacteria. The epithelial cells create the main physical barrier. The
immune system contributes to the gut barrier by sampling luminal antigens.
40
Epithelial M cells internalize antigens; this can cause the activation of
undifferentiated immunocompetent lymphocytes which can then enter into the
intraepithelial region or the mesenteric lymph nodes or systemic circulation
(DeMeo et al. 2002). Mast cells regulate intestinal epithelial migration and
morphology (Groschwitz et al. 2009a).
The mechanisms of gut barrier disruption in acute pancreatitis are poorly
understood. Some intestinal morphological changes have been reported in
pancreatitis patients in the late phase of the disease. The villous height, villous
height/ crypt depth (VH/CD), mast cell index, villous width and area were
significantly reduced in pancreatitis patients who underwent pancreatic necrosis
debridement (Ammori et al. 2002). Eight days after the induction of porcine
pancreatitis, villous oedema, exfoliation and shortening, mucosal erosions and
infiltration of inflammatory cells were seen in the pig’s ileum (Zou et al. 2010).
Disruption of occludin and claudin-1, and the up-regulation of claudin-2,
were all shown in the ileum in the rat model 6 hours after the onset of the disease
(Lutgendorff et al. 2009). Other claudins have not been studied in gut mucosa in
acute pancreatitis.
In a rat model, intestinal epithelial apoptosis was increased and endotoxaemia
occurred 6 hours after, and bacteria translocation 18 hours after, the onset of the
disease (Yasuda et al. 2006). Inhibition of apoptosis in rats had beneficial effects
to gut barrier function and reduced endotoxemia and bacterial translocation 3 to
72 hours after the onset of the disease (Wang et al. 2001b, Nakajima et al. 2007,
Jha et al. 2008).
Endotoxaemia is an early sign of gut barrier dysfunction in pancreatitis. It
was shown to occur 24 hours after hospital admission in pancreatitis patients and
this correlated with the impaired gut permeability (Ammori 2003b). At time of
hospital admission, anti-enterobacterial common antigen titres were significantly
lower in severe pancreatitis patients than in the mild disease or controls
(Kivilaakso et al. 1984).
The cellular cytoskeleton and intercellular tight junctions are dynamic
barriers. Decrease of cellular ATP can cause barrier break down since the process
of barrier maintenance requires energy. This was shown as a mechanism of nonsteroidal anti-inflammaory drugs and tacrolimus interfering with gut barrier
function in rats and humans. Pro-inflammatory cytokines produced locally by
epithelial cells, or reaching the intestinal mucosa from a distant inflammatory
focus, may cause barrier weakening by increasing the nitric oxide production as
41
shown in-vitro. This may be due to the relaxation of the cytoskeleton or
oxidation/ nitration of cytoskeletal proteins (DeMeo et al. 2002).
Tight junctions (TJs)
The intestinal barrier is mainly dependent on the intestinal epithelium (Turner
2006). It is a single-cell layer in which the cells are connected to each other with
tight junctions (TJ), adherens junctions (AJ) and desmosomes. TJs are the most
apical junctions forming a belt-like structure between the apical and basolateral
cell regions around the epithelial and endothelial cells (Forster 2008). TJs are
dynamic multiprotein complexes that are responsible for sealing the intercellular
space and regulating paracellular ionic transport. TJs are fusions where the space
between adjacent cells is eliminated, contrary to AJs or desmosomes where
adjacent cell membranes remain 15 to 20 nm apart ((Groschwitz & Hogan
2009b). The permability of TJs defines the intestinal epithelial barrier function
(Turner 2006). TJs also regulate gene transcription, cell polarity, cell proliferation
and tumor suppression (Schneeberger & Lynch 2004).
Tight junctions are composed of transmembrane proteins that bridge the
apical intercellular space and form a regulated permeability barrier, cytoplasmic
actin binding proteins that serve as links between transmembrane proteins and
actin cytoskeleton and adapters for the recruitment of the cytosolic proteins
required in cell signalling and also a miscellaneous group of cytosolic and nuclear
proteins that coordinate the regulation of paracellular permeability, cell
proliferation, cell polarity and tumor suppression ( Schneeberger & Lynch 2004,
Anderson & Van Itallie 2009).
Tight junctions can be divided into “leaky” and “tight”. TJs in the intestine
are leaky and allow paracellular flux and they are ion and size selective. Size
selectivity differs between the villus and the crypt, with the crypt TJs being leaky
to bigger molecules than the villus TJs (Shen et al. 2009).
Dynamic changes in the tight junction structure are needed during leukocyte
transmigration. Epithelial cells maintain their barrier properties while permitting
neutrophil passage through the paracellular space (Steed et al. 2010).
In acute inflammation of the intestine, the polymorphonuclear leukocytes
(PNM) move from the subepithelial capillaries into and across the epithelium
(Kumar et al. 1982). In epithelial cell monolayers, the PNMs move across the
monolayer by crossing the tight junction. Many crossing PNMs impair the
42
function of the TJ. The impairment appears as decreased TER and increased
permeability to macromolecules (Nash et al. 1987).
More than 40 proteins have been identified in the tight junctions.
Transmembrane proteins include occludin, the families of claudins and junctional
adhesion molecules (JAMs), coxsackie and adenovirus receptor (CAR) and
tricellulin. Cytoplasmic actin binding proteins include zonula occludens proteins1, -2, -3 (ZO-1, -2, -3), membrane-associated guanylate kinase with inverted
orientation proteins-1, -2, -3 (MAGI-1, -2, -3) and multi-PDZ domain protein-1
(MUPP-1) (Schneeberger & Lynch 2004, Tsukita et al. 2008). (Fig. 3)
The expression of junctional proteins in the intestine differs between small
and large intestine, between villus and crypt and between apical, lateral and basal
cell membranes (Groschwitz & Hogan 2009b).
43
44
Fig. 3. Molecular composition of tight junctions. (Groschwitz & Hogan 2009b, published by permission of Elsevier).
Claudins
Claudins are important proteins that regulate tight junction permselectivity (e.g.
size, electrical resistance and ionic charge) (Steed et al. 2010).
There are 27 different claudins known so far (Mineta et al. 2011). The
mammalian claudins are 20 to 34 kDa in size; most of these are 20 to 24 kDa.
Claudins have 4 transmembrane domains, 2 extracellular loops and C and N
cytoplasmic termini (Krause et al. 2008). The C-terminal domain interacts with
PDZ-binding proteins such as ZO-1, -2, -3, membrane-associated guanylate
kinases (MAGUKs), MUPP-1 and Patj and links the claudins to the cytoskeleton
(Lal-Nag & Morin 2009).
Most cells express different claudins. They can form either homophilic (with
the same protein) or heterophilic (between different proteins) interactions between
adjacent cells. Claudins -1, -2, -3, -5, -6, -9, -11, -14 and -19 can form homophilic
interactions and claudin-3 can also form heterophilic interactions with claudin -1,
-2 and -5 (Groschwitz & Hogan 2009b). (Fig. 3)
Different claudins can produce different effects to paracellular permeability.
In in-vitro studies, claudins-2 and -10 have been found to increase the leaking of a
monolayer whereas claudins-1, -4, -5, 7, -8, -11, -14, -15, -16, -18 and -19 make
leaky monolayers tighter (Anderson et al. 2009).
The permeability for solutes smaller than 4Å in radius is dependent on
claudin-based pores that are charge-selective. A second pathway for larger solutes
is based on temporary breaks in the TJs and is not dependent on claudins, lacking
size and charge selectivity (Anderson et al. 2009). This can be enhanced by the
proinflammatory cytokine INF-γ (Watson et al. 2005). Cytoskeletal dynamics
play a role in the non-pore pathway (Hartsock & Nelson 2008).
Claudins -1, -2, -3, -4, -5, -7, -8 and -12 have been detected in the human
intestine (Schulzke et al. 2009, Prasad et al. 2005, Zeissig et al. 2007, Fujita et al.
2008).
The mRNAs of claudins 1 to 12, 14, 15, 17 to 20, 22 and 23 have been
detected in the normal duodena of rats. The mRNA of claudin-13 was seen only
in a rat’s colon (Charoenphandhu et al. 2007). The cDNAs of claudins 7, 8, 12, 13
and 15 are expressed in the duodenum, jejunum, ileum and colon in mice. Their
expression varies along the intestinal tract and also in the subcellular localization
in the intestinal epithelium (Fujita et al. 2006).
45
Disruption of occludin and claudin-1 and the up-regulation of claudin-2 were
observed in the ileal mucosa of rats 6 hours after the onset of pancreatitis
(Lutgendorff et al. 2009). Claudin-5 immunostaining, protein levels and mRNA
decreased in the pancreas at 3, 6 and 12 hours after severe pancreatitis in rats (Xia
et al. 2012). Disruption of occludin and claudin-1 were observed in the pancreata
of mice 10 minutes after caerulein hyperstimulation (Schmitt et al. 2004).
In experimental acute polymicrobial sepsis in mice, the claudin-2 expression
was up-regulated in the colon. Claudins 1, 3, 4, 5 and 8 were displaced from the
apical and lateral membranes of the cell and, in sepsis, they were diffused within
the cells (Li et al. 2009). In polymicrobial sepsis in mice, the absence of COX-2
was shown to regulate the increased permeability and decreased claudin-1
expression (Fredenburgh et al. 2011).
Claudin function is regulated by several mechanisms. Increased
phosphorylation increases paracellular permeability (D'Souza et al. 2005,
D'Souza et al. 2007, Yamauchi et al. 2004). Protein kinase A and C, myosin light
chain kinase (MLCK), rho kinase and ephrin type receptor A1 (EphA1) can all
mediate the phosphorylation of claudins (Findley & Koval 2009). Endocytic
recycling is effective in claudin regulation (Matsuda et al. 2004, Utech et al.
2010). H. pylori and Clostridium perfringens enterotoxins have been shown to
increase claudin internalization (Findley & Koval 2009).
Palmitoylation affects claudin stability (Van Itallie et al. 2005). Transciption
factors Snail (Ikenouchi et al. 2003) and GATA-4 (Escaffit et al. 2005) can bind
to the promoter regions of different claudin genes and affect their expression.
Various growth factors and cytokines can down-regulate claudins both
transcriptionally and post-transcriptionally (Van Itallie & Anderson 2006).
Interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) are found in high
concentrations in the intestinal epithelium in patients with inflammatory bowel
disease (IBD) (MacDonald et al. 1990). These cytokines are found to decrease
barrier function in cultured intestinal epithelial monolayers (Mullin et al. 1992,
Madara & Stafford 1989) and in which they can cause reorganization of claudin-1
and -4. These changes are associated with increased myosin light chain
phosphorylation (Bruewer et al. 2003).
IL-2 knockout mice have ulcerative colitis-like symptoms. In mice lacking
IL-2, the TER was increased and permeability to mannitol was decreased in the
colon mucosa. They had increased expression of the tight junction proteins
occludin and claudin-1, -2, -3 and -5 and they had absorptive dysfunction due to
46
impaired transport protein expression/function while the epithelial barrier was
still intact (Barmeyer et al. 2004).
IL-10 counteracts the effect of proinflammatory cytokines. Mice lacking IL10 expressed colitis and increased levels of TNF-α, IL-1 and IL-6. The ablation of
IL-10 correlates with the altered localization of ZO-1 and claudin-1 (Mazzon E.
2002).
In experimental data from in-vitro colonic epithelial cells, TH2 cytokines IL4 and IL-13 increase intestinal permeability (Zund et al. 1996) by increasing the
expression of claudin-2 and apoptosis (Prasad et al. 2005).
Other tight junction proteins
Occludin has 2 extracellular loops and cytoplasmic C and N termini. The Cterminus interacts with intracellular zonula occludens-1 (ZO-1) which links the
occludin to the cytoskeleton. The extracellular loops and transmembrane domains
regulate paracellular permeability (Groschwitz & Hogan 2009b). Occludin is not
vital for tight junction function since mice lacking occludin showed normally
organized tight junctions (Saitou et al. 1998) with normal transport and barrier
function (Furuse et al. 2002). Occludin has a role in regulating paracellular
permeability (McCarthy et al. 1996) and cellular adhesion (Van Itallie &
Anderson 1997).
Junctional adhesion molecules (JAMs) are single transmembrane proteins
that have 2 immunoglobulin folds in the extracellular domain. Homophilic JAM
interactions regulate the formation of TJs and heterophilic interactions affect
leukocyte-endothelial cell adhesion (Groschwitz & Hogan 2009b).
Tricellulin has four transmembrane domains and is clustered in TJs between
three cells (Tsukita et al. 2008).
ZO-1/2 determines the polymerization of claudins to form TJ strands with
barrier function. ZO-1, -2 and -3 proteins are needed in the fine distribution of
cell polarity proteins (Umeda et al. 2006). ZO-1 and -2 have important roles in
development since mice lacking those proteins died in the embryonic stage
(Katsuno et al. 2008, Xu et al. 2008).
MAGI-1, -2 and -3 proteins express PDZ-domain. MAGI-2 and -3 proteins
interact with growth suppressors. MUPP-1 is a cytosolic PDZ-containing protein
which is expressed only in polarized epithelial cells. It binds to claudins-1 and -8
(Schneeberger & Lynch 2004).
47
The single transmembrane proteins JAMs and CAR cannot form TJ strands
by themselves (Tsukita et al. 2008). CAR is a single transmembrane protein with
two extra-cellular immunoglobin-like domains and a cytoplasmic tail; it functions
as a primary receptor for the coxsackie B virus and adenovirus (Hossain & Hirata
2008).
Adherens junctions (AJ)
AJs are situated between TJs and the most basal junctions, called desmosomes.
AJs and desmosomes link the adjacent cells mechanically. AJs are also important
in the regulation of cellular proliferation, polarization and differentiation
(Groschwitz & Hogan 2009b).
AJs are formed by interactions between transmembrane proteins which are
connected to the cytoskeleton by intracellular proteins. Transcellular E-cadherins
form connections with intracellular catenins. Transcellular nectin interacts with
intracellular afadin (Groschwitz & Hogan 2009b). Cadherin-catenin complexes
link cells together, help maintain cellpolarity and regulate epithelial migration and
proliferation. The decrease of E-cadherin in intestinal epithelium impairs cell to
cell adhesion and causes abnormal proliferation and migration (Perez-Moreno et
al. 2003, Ebnet 2008, Reynolds & Roczniak-Ferguson 2004)In experimental
acute pancreatitis in rats, the E-cadherin protein decreased only moderately in the
pancreas after 2 hours and then remained stable for the whole surveillance time of
12 hours, due to the quick increase of its RNA. E-cadherin-β-catenin is localized
in the basolateral cell membrane but in acute pancreatitis it was dissociated,
internalized and then slowly relocalized. Cytoskeletal actin was proteolytically
cleaved (Lerch et al. 1997). Leukocyte elastase was confirmed to cleave Ecadherin 60 minutes after the disease onset in rats. Further in-vitro analysis
showed that dissociation of cell contacts and the internalization of cleaved Ecadherin to the cytosol followed E-cadherin cleavage (Mayerle et al. 2005).
Secretory granule fusion caused acidification of the pancreatic lumen which led to
disruption of intercellular junctional coupling, measured by movement of
occludin and E-cadherin in pancreatic tissue fragments in-vitro in mice
(Behrendorff et al. 2010).
Serum soluble E-cadherin was found to correlate with disease severity in
human patients during the early phase of acute pancreatitis, at 12 hours or less
(Sewpaul et al. 2009).
48
Loss of normal E-cadherin has been detected around the epithelial ulcerations
in patients with active IBD. Increased gut permeability due to alterations in Ecadherin function may lead to raised local antigen exposure and increased activity
of the intestinal immune system in these IBD patients (Solanas & Batlle 2011).
There are no previous studies concerning E-cadherin function in the intestine in
acute pancreatitis.
1.6.13 Abdominal compartment syndrome (ACS)
Intra-abdominal hypertension (IAH) is reached when intra-abdominal pressure
(IAP) is repeatedly greater than 12 mmHg. ACS is defined as a sustained IAP of
greater than 20 mmHg that is associated with new organ dysfunction / failure.
IAH decreases venous return from the periphery and increases left ventricular
afterload. These phenomena lead to decreased cardiac output, lower arterial
pressure and organ perfusion pressure. Oliguria is usually the first clinical sign.
Hypomotility and swelling of the intestinal walls also occur and, in extreme cases,
ischaemic necrosis of the gut and liver develop (Scheppach 2009).
In severe pancreatitis, fluid resuscitation is needed to maintain cardiac and
renal function. The fluid accumulation in the tissues and abdominal cavity is a
result of the disease but is also aggravated by aggressive crystalloid infusion. It
was reported that IAH has been found in 60 to 80% of SAP patients (Scheppach
2009, De Waele & Leppaniemi 2009). ACS develops in 27% of severe
pancreatitis patients and is associated with a high mortality rate of 50 to 75%.
IAH and ACS are early events in acute pancreatitis. 70% of patients had IAH at
admission to intensive care unit. IAP values tended to be high for 3 to 5 days and
continued to be high in the non-survivors (De Waele et al. 2009).
1.6.14 Systemic inflammatory response syndrome (SIRS) and
Multiorgan dysfunctions syndrome (MODS)
SIRS is manifested when two or more of the following criteria occur: temperature
greater than 38C or less than 36C, heart rate greater than 90 beats/ min,
respiratory rate greater than 20 breaths/ min or PaCO2 greater than 4.3 kPa, WBC
greater than 12 x 109 cells/l, less than 4 x109 cells/l or 10% immature cells. Sepsis
means that SIRS is in response to infection. Severe sepsis criteria are met when
sepsis is associated with organ dysfunction or perfusion abnormalities. MODS is
49
defined by the presence of altered organ function in an acutely ill patient, such
that homeostasis cannot be maintained without intervention (Bone et al. 1992).
There are two hypotheses for the origins of MODS: the microvascular and
cytopathic hypotheses. An exaggerated immune response can cause
hypoperfusion, organ dysfunction and hypoxia leading to MODS. On the other
hand, an elevated immune response may cause NO overproduction, antioxidant
depletion, decreased ATP concentrations and mitochondrial dysfunction leading to
cellular inability to use oxygen, a condition called cytopathic hypoxia or tissue
dysoxia (Castellheim et al. 2009, Antonelli et al. 2007).
The Atlanta classification is criticized for failing to define the severity and
extent of the multi-organ failure. The organ failure is either present or absent. It
discerns no difference as to whether the failure is mild or severe or whether it
includes a single organ or many organs (Acute Pancreatitis Classification
Working Group).
In the new classification, AP is divided into four categories of severity:
pancreatitis is mild if there is no necrosis or organ failure; moderate if necrosis is
sterile and organ failure is only transient; it is considered severe when infected
necrosis or persistent organ failure occurs and it is critical if both co-exist
(Dellinger et al. 2012).
The APACHE II and SOFA scoring systems have high positive prediction
values of the degree of severity of severe acute pancreatitis and can objectify the
responses of patients to intensive care treatment (Beger & Rau 2007).
In acute pancreatitis, only half of patients with pancreatic necrosis will
develop organ failure (Lankisch et al. 2000). The lungs are the most common
organ that fails in acute pancreatitis, then subsequent renal, hepatic,
cardiovascular, digestive, neurologic, coagulation, endocrine or immunologic
failures may follow (Deitch 1992, Kylanpaa et al. 2010). Mortality rates are
relative to which organ has failed: respiratory, renal and hepatic failures lead to
43%, 63% and 83% mortality rates respectively (Halonen et al. 2002). The
number of organs that have failed correlates best with the mortality rate: as the
number of failed organs increases from one to four, the mortality rate increases
from 30% to 100% (Deitch 1992).
Organ failure is an early manifestation in acute severe pancreatitis. It is
already present at admission or will develop during the following 24 hours for 37
to 76% of severe pancreatitis patients (Johnson et al. 2001, Mentula et al. 2003).
Patients with transient (less than 48 hours) organ failure have a better prognosis
than patients with persistent (greater than 48 hours) organ failure ( Johnson &
50
Abu-Hilal 2004a). Patients with pre-existing organ specific conditions may follow
a different course of MODS (Deitch 1992). There is also genetic variation in the
inflammatory response. It seems that polymorphism in the IL-10-1082G gene
plays a role in the susceptibility of SAP patients to septic shock (Zhang et al.
2005) and polymorphism of TNF-α-308A and HSP-70-2G are associated with a
greater risk of severe acute pancreatitis (Balog et al. 2005).
1.7
Clinical course of acute pancreatitis
The first crossroad of the disease occurs 24 to 48 hours after onset, when 20 to
30% of patients take the more severe course. Severe disease is associated with
hypovolemia, fluid sequestration to peripancreatic tissue and the abdominal
cavity. Patients may present pulmonary dysfunction, renal insufficiency, ileus and
circulatory shock (Beger et al. 1997). Intra-abdominal hypertension may develop
(Scheppach 2009). Organ failure (OF) occurs early in the disease course (Johnson
et al. 2001, Isenmann et al. 2001).
In the initial phase, the pancreatic processes evolve dynamically. The
inflammation, oedema and ischaemia can either resolve or lead to necrosis and
liquefaction and/ or the development of fluid collections around the pancreas
(Acute Pancreatitis Classification Working Group).
The second phase of the disease occurs after the second week of the illness.
In this phase the disease either resolves, stabilizes or progresses and the
pancreatic changes occur more slowly (Acute Pancreatitis Classification Working
Group). The second mortality peak occurs after the second week of illness and is
associated with infectious complications such as sepsis and infected pancreatic
necrosis. Patients with organ failure and infected pancreatic necrosis have a
significantly higher risk of death than patients with either organ failure or infected
pancreatic necrosis (Petrov et al. 2010).
Infection is time-dependent. 25% of the contamination occurs during the first
week of the illness, 45% by the second week and 60% by the third week of
necrotizing pancreatitis (Beger et al. 1997). Pancreatic abscesses occur in 3% of
patients as a late complication after necrotizing pancreatitis; this is associated
with a 6.5% mortality rate (Schoenberg et al. 1995).
Pancreatic ductal necrosis can result in the disconnection of the main
pancreatic duct, as a result of which there is a lack of ductal connection between a
viable pancreatic segment and the gastrointestinal tract. The exocrine output of
the viable pancreatic segment induces fistulas or persistent collections. The
51
incidence of disconnected ducts is not known but may vary between 16 and 47%
(Sandrasegaran et al. 2007, Lawrence et al. 2008). Surgical necrosectomy and
percutaneous catheter drainage result in persisting pancreatic fistulas in 17 to 76%
of cases. Pancreatic fistula closure is achieved in 75% of conservatively treated
patients with a median time of 120 days. Endoscopic transpapillary stenting
increases the fistula closure rate to 84% with the median time to closure being 71
days (Bakker et al. 2011).
Peri-pancreatic fluid collections persisting more than 4 weeks are termed
pseudocysts. Pseudocysts are encapsulated by fibrous and granulation tissue
(Bharwani et al. 2011). 6% of patients develop pseudocysts. Pseudocysts less than
6 cm in diameter and having no connection with the pancreatic duct will have a
40% chance of spontaneous resolution (Yeo et al. 1990).
Pancreatic functional and morphologic abnormalities will last for years after
the disease and they are dependent on the degree of necrosis. Exocrine function
ameliorates 12 to14 months after severe pancreatitis. The glucose intolerance is
however, likely to worsen over time (Beger HG. 1997, Sand & Nordback 2009a).
1.8
Experimental models of acute pancreatitis
It is often not possible to examine the pathogenenesis of acute pancreatitis in
human patients since the methods are invasive and would increase complications
in already seriously ill patients. Consequently, several animal models of
experimental acute pancreatitis have been introduced. A model should be
reproducible, cheap and simple. It should mimic the human disease and have a
similar response to treatment. However, there are no exquisite models at the
moment. Most factors employed to cause experimental pancreatitis are not known
causes in the human disease. The human disease can also vary widely in severity
regardless of the etiology and the human disease has different phases: an initiation
phase, early phase, middle phase and late phase. The experimental models can
typically mimic the initiation and early phases but if the animal survives the early
phase, it usually survives thereafter (Rattner 1996). The disease course in small
rodents is faster than in humans (van Minnen et al. 2007).
Mice and rats are the most widely used animals in experimental studies
concerning acute pancreatitis (Banerjee et al. 1994). With rodents it is possible to
perform experiments using at least two dissimilar experimental models. This is
beneficial in reducing the effect of model-specific responses to the results
(Laukkarinen et al. 2007). Transgenic technology has led to the availability of
52
modified mouse strains in mechanistic studies of acute pancreatitis (Wan et al.
2012). Cell and organ culture models will reduce the need for experimental
animal studies. Especially, cell culture models provide an in-vitro setting in which
cellular responses can be monitored under accurately defined conditions in the
study of the mechanisms of acute pancreatitis (Bläuer et al. 2011).
Larger animals, such as pigs, dogs, rabbits, goats and opossums, have also
been used in studies of experimental acute pancreatitis (Banerjee et al. 1994).
Although the tendency is to minimize the use of bigger animals, they are still
needed for studies in which the size of animal is crucial, such as studies using
invasive monitoring.
Experimental models can be duct injection or duct ligation induced, diet
induced, secretagogue induced, microvascularly induced or immunological
(Banerjee et al. 1994). The most commonly used models are caerulein
intravenously induced, choline deficient ethionine supplemented (CDE), diet
induced and duct injection induced forms of pancreatitis (Chan & Leung 2007).
1.
Infusion of substances into the pancreatic duct
Different concentrations of bile or bile salts are used to obtain moderate to
severe acute pancreatitis (Lankisch & Ihse 1987). Duct injection causes rapidly
and reliably severe necrotizing pancreatitis to rats or other larger animals, e.g.
pigs or dogs. The model is well-established, repeatable and clinically relevant. It
remains the most prevalent model in studies examining the pathogenesis and
therapies since it is similar to the human disease. Intraductal injection of the bile
acid taurocholate or glycodeoxycholic acid in low concentration leads to a disease
mimicking oedematous biliary pancreatitis while a high concentration of
taurocholate leads to necrotizing hemorrhagic pancreatitis (Chan & Leung 2007).
3 to 6% taurocholate induces severe pancreatitis in rats. Pigs are less susceptible
to injury and require higher a concentration, of 15% taurocholate (Kinnala et al.
1999). The duct injection model induces multiple organ failure involving the lung,
kidney, liver, intestine and brain (Chan & Leung 2007). The impact mechanism of
duct injection is partially due to the increased hydrostatic pressure, since
intraductal injection of saline has been reported to induce mild pancreatitis in rats
and pigs (Lichtenstein et al. 2000, Kinnala et al. 1999).
The limitation of the model is the need for a surgical procedure to place the
catheter into the pancreatic duct (Banerjee et al. 1994). It was demonstrated that
biliopancreatic duct cannulation does not increase the risk of pancreatic infections
53
in rats (Foitzik et al. 1994b); however, intestinal handling during surgery can
potentially affect the mucosa barrier function (van Minnen et al. 2007).
2.
Ligation of the pancreatic duct
Duct obstruction induced acute pancreatitis imitates the etiology of gallstone
or tumour induced acute pancreatitis in a clinical setting (Mooren et al. 2003).
Ligation of the pancreatic duct in rodents can also be combined with the
administration of chemicals such as cholecystokinin, caerulein or secretin. The
acute pancreatitis reverses when the duct obstruction is removed, allowing the
study of treatment intervals (Chan & Leung 2007).
3.
Dietary pancreatitis
CDE-diet induced acute pancreatitis was first reported in 1975 (Lombardi et
al. 1975). The CDE-diet causes hemorrhagic pancreatitis with massive fat
necrosis to female rats. The mortality rate and the degree of pancreatic damage
can be controlled in rats (Steer & Meldolesi 1987) but mice suffer 100% mortality
when fed this diet for four days (Lombardi et al. 1975). The exact mechanism of
how the CDE-diet causes acute pancreatitis remains unclear. The CDE-diet
directly affects the liver and brain, causing formation of fatty liver and inhibition
of brain protein synthesis. The degree of pancreatic injury is mild and the
incidence of pancreatic infection is low (3 to 8%). This model is suitable for
studying the pathophysiology of acute pancreatitis but not appropriate for
studying SIRS, MODS or pancreatic infections. The small size of the animals is
also a limitation in this model (Chan & Leung 2007, van Minnen et al. 2007).
4.
Secretagogue induced pancreatitis
The cholecystokinin analogue caerulein induces proteolytic enzyme
secretion, pancreatic acinar autolysis and oedematous pancreatitis resembling the
human disease histologically. It is possible to control the degree of injury but the
injury is self-limiting. It is suitable for rats, mice and dogs. In mice and rats it
produces an ARDS-like lung injury (Guice et al. 1988). It can also be
administered to isolated acini or exocrine cells, enabling in-vitro studies (Chan &
Leung 2007). The cerulein model is useful when studying early oedematous
pancreatitis in general terms but the pathogenesis is different to the human disease
(Banerjee et al. 1989). This model is associated with reduced bowel motility,
increased small bowel bacterial overgrowth and increased bacterial translocation
and thus interferes with the results of translocation studies (van Minnen et al.
2007).
54
Intraperitoneally-given arginine induced acute pancreatitis is a less invasive
acute pancreatitis model in rats. The severity of the acute pancreatitis is dependent
on the dose of the arginine (Ishiwata et al. 2006).
5.
Boston model
A combination of bile salt infusion and hyperstimulation with caerulein in
rats is referred to as the “Boston model”. It has high degree of similarity to the
human disease, mimicking human pancreatic histological changes, the spectrum
of pathogenic micro-organisms in the tissue, MODS and delayed mortality
(Schmidt et al. 1992).
6.
Closed duodenal loop-induced pancreatitis
One theory of the etiology of acute pancreatitis is that of duodenal reflux
induced acute pancreatitis. In closed duodenal loop-induced experimental
pancreatitis, a “blind loop” is created by dividing the duodenum distal to pylorus
and then again distal to pancreas head. A gastrojejunostomy is also made. The
exact initiating factor in closed duodenal loop-induced pancreatitis is uncertain,
decreasing the clinical relevance. The model is suitable for larger animals but the
complicated and invasive surgical procedure required has decreased the
popularity of this model (Chan & Leung 2007).
7.
Pancreatitis caused by immune reactions
After intravenous/subcutaneous sensitization to ovalbumin, acute pancreatitis
has been induced in rabbits by a pancreatic intraductal injection of ovalbumin.
Additionally, intraductal and intravenous injections of meningococcus and E.coli
endotoxin will produce acute pancreatitis. Injection of rabbit serum
intraperitoneally or intraductally in rats causes acute necrotizing pancreatitis.
These models are associated with a high rate of early mortality and non-specific
generalized immunological response in other organs; immunological models are
not commonly used due to these limitations (Banerjee et al. 1994).
8.
Vascular-induced pancreatitis
Impairment of pancreatic inflow, outflow or microcirculation leads to
pancreatitis in many animals and in humans. Creating a low-flow state by
inducing hypovolemic shock decreases the inflow. This mimics the pancreatitis
associated with extensive surgery, such as cardiac surgery. The occlusion of the
superior pancreaticoduodenal artery results in necrotizing pancreatitis but this
etiology is rare in the human disease. Venous ligation and venous injection of
microspheres also lead to acute experimental pancreatitis. A disadvantage of these
55
models is the necessary surgical trauma and often the need for bigger animals. In
hypovolemic shock-induced pancreatitis, the damage is not limited to the
pancreas and thus it is not optimal for studying pancreatitis associated MODS.
Disturbance of pancreatic microcirculation results in low repeatability (Chan &
Leung 2007).
56
2
Aims of the study
This thesis was undertaken in order to ascertain answers to the following
questions:
1.
2.
3.
4.
What happens in pancreatic microcirculation during the early phase of acute
experimental pancreatitis and do the microcirculatory changes differ between
oedematous and necrotizing pancreatitis? Is there some difference between
oedematous and necrotizing experimental acute pancreatitis in the expression
of claudins-2, -3, -4, -5 and -7 and the rate of apoptosis in the pancreas
during 180 minutes of surveillance?
Does bacterial translocation to the portal vein blood or mesenteric lymph
nodes occur during 360 minutes follow-up, after the onset of acute
experimental oedematous and necrotizing pancreatitis? Does the intestinal
expression of tight junction proteins (claudins-2, -3, -4, -5 and -7), apoptosis
rate or proliferation rate explain the possible translocation? Are there changes
in the basic histology of the jejunum or colon in oedematous or necrotizing
pancreatitis?
Which cytokines are released from the pancreas to portal venous blood in the
early phase (360 minutes) of acute experimental oedematous and necrotizing
pancreatitis and which of those cytokines are correlated with the more severe
form of the disease?
Are there changes in the ultrastructure of the epithelium of the jejunum and
colon in acute oedematous and necrotizing pancreatitis during 540 minutes of
follow-up? Are there changes in the expression of E-cadherin and β-catenin
proteins that would explain the structural changes in the adherens junctions?
57
58
3
Animals and methods
3.1
The Porcine model
The acute pancreatitis porcine model has been described in 1982 in Sweden. In
this model, acute necrotizing pancreatitis is induced in pigs by the retrograde
injection of taurocholic acid into the pancreatic duct (Ruud et al. 1982). Acute
oedematous pancreatitis was first induced by the intraductal retrograde injection
of autologous bile or the injection of free fatty acid (FFA) into the pancreatic
artery (Puolakkainen et al. 1989, Vollmar et al. 1989); since then, intraductal
retrograde injection of saline has been shown to cause acute oedematous
pancreatitis (Kinnala et al. 1999).
The porcine model was chosen because porcine anatomy and physiology
resembles human anatomy and physiology better than that of rats or mice. The
porcine animal size enables the cannulations and measurements used in these
studies.
The retrograde duct injection model reliably produces oedematous and
necrotizing pancreatitis, which resembles the human disease. This model enables
comparison between mild oedematous and severe necrotizing pancreatitis.
3.2
Test animals and preoprative management
The animals involved in these studies were juvenile (aged 8 to 10 weeks) pigs
from a native stock with a median weight of 28.3 kg (24.95 to 30.65 kg).
All animals received humane care in accordance with the Principles of
Laboratory Animal Care formulated by the National Society for Medical Research
and the Guide for the Care and Use of Laboratory Animals prepared by the
Institute of Laboratory Animal Resources, National Research Council (published
by National Academy Press, revised in 1996). The study was approved by the
National Animal Ethics Committee in Finland.
The animals were provided by a nearby piggery and they were allowed to
adapt to the new environment in the Laboratory Animal Centre for one week
before the experiment.
59
3.3
Anaesthesia, haemodynamic and temperature monitoring
The same anaesthesia method was applied in all study groups. The animals were
fasted for 24 hours before the experiment with free access to water. Pigs were
sedated with ketamine hydrochloride (350 mg i.m.) and midazolam (45 mg i.m.).
Animals were placed in a supine position on a heater mattress and an electric
heater was used to maintain body temperature.
A peripheral intravenous line was inserted into the veins of both ears for the
administration of drugs and fluids. An arterial line was positioned in the right
femoral artery and a pulmonary artery catheter (CritiCath, 7 Fr; Ohmeda GmBH,
Erlangen, Germany) was placed through the right femoral vein to allow blood
sampling, cardiac filling pressure (central venous pressure, pulmonary artery
pressure and capillary wedge pressure), cardiac output monitoring and central
temperature measurement. A temperature probe was placed in the rectum and a 10
Fr catheter was placed in the urinary bladder to monitor urinary output.
Anaesthesia was induced with fentanyl (25 μg/kg i.v.) and pancuronium (0.2
mg/kg i.v) and maintained with continuous infusions of fentanyl (25 μg/kg/h),
midazolam (0.25 mg/kg/h), pancuronium (0.2 mg/kg/h) and inhaled isoflurane
(0.5%) in every study group throughout the experiment. The pigs were intubated.
Positive pressure ventilation was adjusted according to blood gas analyses,
targeting to maintain normocapnia (PaCO2 4.5-5.5 kPa) and normal arterial blood
oxygen tension (PaO2 >13.3 kPa) with 50% inspired oxygen, 8 to13 ml/kg lung
tidal volume and 5 H2O positive end expiratory pressure.
Normal haemodynamic parameters were targeted during the experiment. In
Study I, fluid balance was maintained with 0.9% saline (NaCl) 5 ml/h/kg. In
Studies II to IV, saline (NaCl 0.9%) was infused at a rate of 10 ml/h/kg
throughout the whole experiment and if the mean systolic arterial pressure
dropped below 60 mmHg or pulmonary capillary wedge pressure fell below 5
mmHg, a bolus dose of 100 ml of Ringer´s acetate and Voluven (Fresenius Kabi,
Finland) at a ratio of 1:1 was administered. In studies II to IV, blood glucose
levels were targeted between 5 and 7 mol/l and a 30% solution of glucose was
infused (1 to 1.5 mg/kg) to maintain blood glucose level within the targeted
range.
60
3.4
Experimental protocols
A laparotomy was performed. In all studies, animals were randomly allocated to
the study groups. In study I, they were randomized to oedematous and necrotizing
pancreatitis groups and in Studies II to IV they were randomized to oedematous
and necrotizing pancreatitis and control groups.
To cause a necrotizing or oedematous pancreatitis, the pancreatic duct was
cannulated with a 3Fr urether cannula. The cannula was placed and left 5 cm deep
in the duct and attached to the wall of the duodenum. Duodenotomy was closed
by continuous suture. For the control animals, duodenotomies were not done and
their pancreatic ducts were not cannulated.
In Study I, tissue samples from the left lobe of the liver, duodenum, left
kidney and caecum were taken but not analysed. The caecum and duodenum were
sutured after biopsy. The duodenal loop with the head of the pancreas was
carefully fixed on a rigid stand and a tissue sample from the corpus of the
pancreas was harvested for histological analysis. A microdialysis catheter was
inserted in the middle of the head of the pancreas.
In Studies II to IV, tissue samples were taken from the distal pancreas, the left
lobe of the liver, the jejunum (30cm proximally from the ileocaecal junction) and
the colon (60 cm distal to the ileocaecal junction). In study II, a mesenteric lymph
node was harvested as a sample from the ileocaecal corner. Liver samples were
not analysed in this study. In study III only the pancreas samples were analysed.
The openings of the jejunum and the colon were closed with sutures.
A 16 G catheter (central venous catheter; Secalon Seldy, Singapore) was
placed in the portal vein to allow blood sampling.
The animals were left to stabilize for 30 minutes. Thereafter, baseline
haemodynamic measurements and blood samples were obtained and in Study I,
the baseline microdialysis sample was collected and intravital microscopy (IVM)
performed.
Necrotizing pancreatitis was induced with a 20% sodium-taurocholate
injection 1 ml/kg (Sigma, Germany) and oedematous pancreatitis with a 0.9%
NaCl 1 ml/kg injection. Both retrograde injections were given into the pancreatic
duct within 30 minutes via an automatic infusion pump.
In Study I, each animal was monitored for 180 minutes, in Studies II and III
for 360 minutes and in Study IV for 540 minutes.
Thereafter, in Study I futher samples from the left lobe of the liver,
duodenum, left kidney and caecum were collected and whole pancreas was
61
harvested as a sample. In Studies II to IV, further samples from the liver, the
jejunum and the colon were collected and the whole pancreas was harvested as a
sample. In study II, mesenteric lymph node samples were harvested after 120, 240
and 360 minutes. In Study II, the abdomen was closed and only temporarily
opened during the lymph node harvesting.
At the end of the experiment, each animal was euthanized with pentobarbital.
3.5
Intravital fluorescence microscopy
An epifluorescence microscope (Model MZ FLIII, Leica; Heerbrugg,
Switzerland) containing a 100 W mercury gas discharge lamp with rapid filter
exchanger was placed over the head of pancreas. The duodenal loop with the head
of the pancreas was fixed on a rigid stand. The microscope images from the
charge-coupled device video camera (Dage-300-RC; Dage-MTI, Michigan City,
IN) were time-stamped using a time-code generator (VTG-33, For-A) and
transferred to a high-resolution 12-inch monitor (Dage HR-1000; Dage-MTI) for
videotaping. A Scion LG-3 frame grabber card (Scion Corp, Frederick, MD),
along with a computer-assisted image analysis system (NIH Image, National
Institutes of Health, Bethesda, MD) was used for subsequent offline analysis with
the final image magnification being ×400. Circulating leukocytes were labeled
with 2 mL of 0.2% rhodamine (Sigma Chemical, St. Louis, MO). Arterial and
venous diameters were measured from three different locations at each time point.
Intravital fluorescence microscopy was performed at baseline and at 5, 10, 20, 30,
60, 90, 120, 150 and 180 minutes. The duration of the epi-illumination of the
pancreatic tissue was limited to less than 90 seconds and was shut off between
video recordings to avoid thermal injury. The ventilator was shut off and a bolus
injection of pancuronium was given for the recording to avoid disturbances
caused by small movements.
3.6
Microdialysis
The microdialysis catheter (30mm CMA 71, High Cut-Off Brain Microdialysis
Catheter; CMA Microdialysis, Stockholm, Sweden) was placed in the middle of
the pancreatic tissue in the head of the pancreas. The microdialysis catheter was
connected to a 2.5-mL syringe placed in a microinfusion pump (CMA 107; CMA
Microdialysis) and perfused with a Ringers solution 5 μL/min (Perfusion Fluid;
62
CMA Microdialysis). Samples were collected every 30 minutes. The amounts of
protein, albumin and potassium were measured from the perfusate.
3.7
Blood samples, portal vein blood cultures and mesenteric
lymph node bacterial cultures
In all studies, systemic arterial and venous blood samples were obtained to
determine haemoglobin, haematocrit, white blood cell differential count, platelet
count, creatinine, amylase, lipase, blood gases (pH, PCO2, PO2, SaO2, base excess),
sodium, potassium, calcium and lactate.
In Study II, portal venous blood samples and mesenteric lymph nodes were
taken for bacterial culture. Both portal venous blood samples and mesenteric
lymph node samples were cultured within 15 minutes. Lymph nodes were crushed
before culturing. Both portal vein blood and mesenteric lymph node samples were
cultured on blood, anaerobic and chocolate agars for 4 days. The amounts of the
bacteria were estimated macroscopically, one bacteria colony representing 103
bacteria.
3.8
Tissue samples
In Study I, a baseline sample from the corpus of the pancreas was harvested
before causing pancreatitis. After 180 minutes, the whole pancreas was obtained.
It was fixed in neutral buffered formalin for 3 days after which, sections from the
head, corpus and cauda were taken as samples. The samples were embedded in
paraffin and 5-µm thick sections were cut for later use in immunohistochemistry
and TUNEL labeling. Additionally, haematoxylin–eosin-stained sections were
made from the samples.
In study II, samples from the pancreas, the jejunum and the colon were
harvested before causing pancreatitis and after 360 minutes. The samples were
fixed in neutral buffered formalin for 3 days, embedded in paraffin and cut into 5µm thick sections for use in immunohistochemistry, TUNEL labelling and
haematoxylin–eosin stainings. Epithelial cell height in the jejunum, mucosal
thickness in the colon, villus height, crypt depth, thickness of both submucosa and
serosa exudate in the jejunum and colon were all measured from the
haematoxylin-eosin-stained tissue sections. The haematoxylin–eosin-stained
sections of the pancreatic samples were analyzed to assess the severity of the
pancreatitis by a blinded investigator.
63
In Study IV, samples from the pancreas, the jejunum and the colon were
collected before causing pancreatitis and after 540 minutes. The samples were
fixed in neutral buffered formalin for 3 days, embedded in paraffin and cut into 5µm thick sections for use in immunohistochemistry and haematoxylin–eosin
stainings.
3.9
Immunohistochemistry and Histopathological analysis
In Studies I and II, the primary antibodies that were used for
immunohistochemistry were all purchased from Zymed Laboratories Inc. (South
San Francisco, CA) and designed for use in formalin-fixed paraffin-embedded
tissues. They included monoclonal mouse claudin-2 antibody, polyclonal rabbit
anticlaudin 3, monoclonal mouse anticlaudin 4, monoclonal mouse anticlaudin 5
and polyclonal rabbit anticlaudin 7. Before application of the primary antibodies,
the sections were heated in a microwave oven in 10 mM citrate buffer, pH 6.0, for
10 minutes. After 60 minutes incubation with the primary antibody (dilution 1:50
for anticlaudins 2, 3, 4, 5, and 7), a biotinylated secondary anti-rabbit or antimouse antibody and Histostain-SP kit (Zymed Laboratories) was used. For all the
immunohistochemistry, the colour was developed by diaminobenzidine and,
subsequently, the sections were lightly counterstained with haematoxylin and
mounted with Eukitt (Kindler, Freiburg, Germany).
In Study IV the primary antibodies that were used for immunohistochemistry
were all purchased from BD Biosciences (San Jose, CA, USA) and designed for
use in formalin-fixed paraffin-embedded tissues. They included E-cadherin and βcatenin. Before application of the primary antibodies, the sections were heated in
a microwave oven in 10 mM citrate buffer, pH 6.0, for 29 minutes. After 60
minutes incubation with the primary antibody, a biotinylated secondary anti-rabbit
or anti-mouse antibody and polymer based detection (Envision, Dako Glostrup,
Denmark) was used. For all the immunohistochemistry, the colour was developed
by diaminobenzidine and, subsequently, the sections were lightly counterstained
with haematoxylin.
Negative control stains were carried out by substituting the primary
antibodies with phosphate buffered saline. For positive controls, specimens from
the control series were assessed for a characteristic expression of Ki-67 in the
proliferating epithelial cell nuclei in the crypt bases and in the germinal centers of
the mucosal lymphoid follicles. In a corresponding way, characteristic
membranous expression of claudin-2, -3, -4, 5 and -7, E-cadherin and β-catenin in
64
the columnar epithelial cells, as reported in the literature, was used as a positive
for these antigens (Prasad et al. 2005, Zeissig et al. 2007, Kucharzik et al. 2001).
For TUNEL stainings, a mucosal lymphoid follicle with germinal centers was
used as a positive control.
In Study I, the immunoreactivity in the pancreas was assessed by two
investigators (SM and YS). The extent of membranous immunostaining in both
acinar and ductal cells was evaluated for all claudins in different sections of the
pancreas both qualitatively and quantitatively.
In Study II, the immunoreactivity in the jejunum and colon was assessed
quantitatively. Quantification was carried out by using a computer program
(Scientific Image Analysis; MCID-M4 3.0 Rev 1.1). Mucosal immunoreactivity
was analyzed from 10 villi and cryptae from each sample. Colour intensity and
saturation were measured. Furthermore, the sizes of the stained area (total target
area) and the scanned area were measured, as well as their proportional area. The
numbers of Ki-67 positive cells were counted from 10 villi and cryptae in each
sample. The positive cells from the upper half of each villus in the jejunum and
the upper half of the mucosa in the colon were counted separately.
In Study IV, the mucosal immunoreactivity was analyzed from 10 villi and
cryptae from each sample. Colour intensity and saturation were measured.
Further, the extent of the stained area (total target area) and the scanned area were
measured, as well as their proportional areas. In the analysis, the upper and lower
halves (50%) of the mucosa were measured separately.
3.10 Apoptosis
The rate of epithelial cell apoptosis was determined by TUNEL-labelling. The
sections, after dewaxing in xylene and rehydration in ethanol, were incubated
with Proteinase K (20 mg/mL) for 15 minutes. Apoptotic cells were demonstrated
using an ApopTag Peroxidase In-Situ Apoptosis Detection Kit S7100 (Chemicon
International, USA) according to the manufacturer’s instructions. A cell was
defined apoptotic if the whole nuclear area gave a positive brown-coloured
reaction. Only cells showing positive labelling and clear morphological signs of
apoptosis, i.e. nuclear condensation, cell shrinkage, cytoplasmic budding to form
membrane-bound fragments and detachment from surrounding cells, were
considered positive. For quantitative analysis, 10 fields of ×40-magnification of
each sample were examined, and the results were reported as the number of
apoptotic cells per field.
65
3.11 Cytokine assays
The cytokine levels were analyzed with Bio-Plex Pro Human Cytokine Assay
(Bio-Rad Laboratories Inc. Catalog # M500KCAFOY, CA, US) and Milliplex
Human Cytokine/Chemokine Magnetic Panel (Millipore, Catalog #
MPXHCYTOMAG-60K, Millipore Corporation, Billerica MA, US) utilizing a
Bio-Plex instrument based on Luminex xMAP Technology (Bio-Rad Laboratories
Inc., CA, US). The measurement was performed according to manufacturer’s
instructions (Myhrstad et al. 2011). Assay conditions were standardized and
optimized to ensure optimal reproducibility of the assays. The plasma samples
were centrifuged for 10 min at 13300 g prior to the analysis and diluted 1:1
(Milliplex) or 1:2 (Bio-Plex) in an appropriate sample matrix. The results were
automatically calculated with Bio-Plex Manager Software with five-parameter
logistic equations. High sensitivity range standard settings were utilized for the
Bio-Plex assay. The concentration of PDGF, IL-1, IL-1ra, IL-2, IL-4, IL-5, IL-6,
IL-7, IL-8, Il-9, IL-12, IL-13, IL-15, IL-17, eotaxin, fibroblast growth factor
(FGF), granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage
colony-stimulating factor (GMCSF), IFN- γ, interferon gamma-induced protein
10 (IP-10), MCP, MIP-1α, regulated upon activation normal T-cell expressed and
secreted protein (RANTES), TNF-α and vascular endothelial growth factor
(VEGF) were all measured from portal venous blood samples. However, only
PDGF, IL-2, IL-4, IL-6, IL-7, IL-8, IL-9, eotaxin, IFN- γ, MCP-1 and MIP-1α
were measurable from pig’s blood.
In order to compare the cytokine concentrations in portal venous blood to
concentrations in systemic blood we measured the cytokines eotaxin and MCP-1,
since they were inflammatory cytokines which increased during the course of the
pancreatitis and were in a measureable range in our assays.
3.12 Transmission electron microscopy
Specimens from the epithelium of the jejunum and colon and from the mesentery
of the jejunum were fixed in a 1 % glutaraldehyde / 4% formaldehyde mixture in
0.1 M phosphate buffer. They were postfixed in 1% osmiumtetroxide, dehydrated
in acetone and embedded in Epon LX 112 (Ladd Research Industries, Williston,
Vermont, USA). Thin sections were cut using a Leica Ultracut UCT
ultramicrotome, stained in uranyl acetate and lead citrate and examined in a
Philips CM100 transmission electron microscope.
66
3.13 Statistical analysis
Statistical analysis was performed using SPSS (SPSS, version 14.0; SPSS Inc,
Chicago, IL) and SAS (version 9.1.3; SAS Institute Inc., Cary, NC) statistical
software. Continuous and ordinal variables are expressed as the median with 25th
and 75th percentiles. The t test was used to compare the study groups. The
analysis of variance (ANOVA) was used when comparing single outcomes as
well as outcomes with two measurements. In the latter case, the difference (first to
second measurement) was calculated and then the between group comparison was
performed for the difference. If the ANOVA showed a significant difference in the
between group, the post-hoc comparisons were calculated using Tukey’s HSD
(honestly significant difference). The mixed-model approach was used to analyze
repeated measurements for variables with more than two measurements. The
reported P values are as follows: P between groups (Pg), indicating a level of
difference between the groups; P time*group (Pt*g), indicating the group time
interaction and (Pt), indicating change over measurement points. Two-tailed
significance levels are reported, where a P<0.05 was considered statistically
significant.
67
68
4
Results
4.1
Animals excluded from data analysis
In total 77 pigs were operated on. Pilot studies were necessary in all study setups: in Study I, to create the model and to optimize the utilization of the intravital
microscope (IVM) and microdialysis. In Studies II to IV, the pilots were needed
to optimize the model with regard to fluid management and experimental
protocols. In Studies II and III, pilots were used in testing IVM and microdialysis
in the analysis of the mesenteric and intestinal microcirculation. Altogether, 52
animals were included in the final data analysis. The reasons for exclusions are
presented in Table 1. The number of animals in each series was: 13 in Study I, 15
in Study II (and III) and 24 in Study IV.
Table 1. The number of animals excluded from the data analysis.
Indication for exclusion
Pilot studies
Study I
Study II+III
Study IV
14
6
2
Intestinal ischemia due to
1
mechanical strangulation
Bleeding
4.2
1
1
Comparability of the study groups
The weight of the pigs did not differ between the study groups and the control
group in any study. The baseline measurements indicated no significant
differences between experimental groups regarding baseline haemodynamics,
blood gases, haemoglobin, haematocrit, white blood cell differential count,
platelet count, creatinine, amylase, lipase, sodium, potassium, calcium and lactate.
4.3
Haemodynamic variables
In Study I, the heart rate tended to increase in both groups, although the change
was statistically non-significant. In both groups, the systolic, diastolic and mean
arterial blood pressure decreased (Pt = 0.044; Pt = 0.052; Pt = 0.011,
respectively). These changes lead to a diminishing of cardiac output (Pt = 0.005).
As a consequence of these events, pulmonary artery systolic and diastolic blood
pressures decreased in both study groups (Pt = 0.011 and Pt = 0.0002,
69
respectively). Central venous pressure and pulmonary capillary wedge pressure
did not alter in either group.
Rectal and pulmonary artery temperatures increased in necrotizing
pancreatitis, while they decreased in oedematous pancreatitis. The normal body
temperature for pigs is between 37oC and 38oC, however, and therefore both
groups were considered normothermic towards the end of the experiment.
In Study II, the groups did not differ in haemodynamic variables.
4.4
Blood samples
In Study I, the levels of serum lipase rose in both groups, indicating the
development of pancreatitis (Pt = 0.001). The levels of amylase rose only in the
necrotizing pancreatitis group. The potassium level increased in both groups (Pt =
<0.001). Glucose, calcium and lactate levels tended to decrease in both groups (Pt
= 0.008; Pt = 0.013; Pt = 0.011 respectively). The levels of haemoglobin,
haematocrit, creatinine, and blood gases remained unchanged in both groups.
In Studies II and III, haemoglobin concentration and haematocrit increased in
all groups, indicating slight hypovolemia due to the experimental procedure. The
amounts of liquids infused did not differ between the study groups. Arterial and
venous pH decreased in all groups (P<0.001). Arterial and venous pCO2 increased
(P=0.02, P=0.03) and arterial pO2 (P=0.02) decreased in all groups and there were
no differences between the study groups.
There was elevation of blood lipase (P=0.004) and amylase (P<0.001) levels
in the necrotizing pancreatitis group, indicating the emergence of pancreatitis as
compared to the control group and a more severe disease as compared to the
oedematous pancreatitis group. In the oedematous pancreatitis group, venous
blood lipase increased but amylase did not. The control group´s lipase and
amylase levels remained the same.
In Study IV, there was elevation of blood lipase levels in the necrotizing
pancreatitis group indicating the emergence of pancreatitis as compared to the
control group and a more severe disease as compared to the oedematous
pancreatitis group.
4.5
Blood cells
In Study I, the white blood cell count increased in the necrotizing pancreatitis
group (Pt = 0.009). The amount of neutrophils and monocytes increased in both
70
groups (Pt = 0.008; Pt < 0.001). The amounts of lymphocytes (Pt < 0.001) and
platelets (Pt < 0.001) decreased in both study groups. Plateletcrit also diminished
in both groups (P = 0.017). The blood cell changes were seen as soon as the first
measurement 30 minutes after the induction of pancreatitis. There were no
changes in the amount of red blood cells and no significant changes in the amount
of eosinophiles and basophiles between the two groups. However, there was a
decrease in the amount of eosinophils and an increase in the amount of basophils
in both groups.
In Studies II and III, the white blood cell count increased in the necrotizing
pancreatitis and control groups. The amount of neutrophils increased in all groups
(P<0.001) (Fig 4). The lymphocyte and eosinophil counts decreased in all groups
(P<0.001, P<0.001) (Fig 5-6). The monocyte count increased only in the
necrotizing pancreatitis group (Fig 7).
Fig. 4. The venous blood neutrophil count in Study III.
71
Fig. 5. The venous blood lymphocyte count in Study III.
Fig. 6. The venous blood eosinophil count in Study III.
72
Fig. 7. The venous blood monocyte count in Study III.
73
4.6
Portal vein cytokines
In study III, PDGF increased only in the necrotizing pancreatitis group. The
increase was already visible after 2 hours; beyond 2 hours, the concentration
remained the same (Fig8).
After 2 hours there was also a slight increase in IL-6, but only in the
necrotizing pancreatitis group (Fig 9).
IL-8 and eotaxin increased both in the oedematous and necrotizing
pancreatitis groups. Their concentration peaked after 2 hours and decreased
thereafter (Fig 10, 11).
MCP-1 increased during the entire follow-up period in all groups. However,
these changes were not statistically significant between the study groups (Fig 12).
IL-9, IL-4, MIP-1α and IFN- γ concentrations did not change.
There was no significant difference in the median differences of eotaxin and
MCP-1between portal venous blood and pulmonary arterial blood (Fig 13).
Fig. 8. The concentration of platelet-derived growth factor in portal venous blood.
74
Fig. 9. The concentration of interleukin-6 in portal venous blood.
Fig. 10. The concentration of interleukin-8 in portal venous blood.
75
Fig. 11. The concentration of eotaxin in portal venous blood.
Fig. 12. The concentration of monocyte chemotactic protein in portal venous blood.
76
Fig. 13. Median differences between portal venous blood and pulmonary arterial blood
measures of eotaxin and MCP-1. The horizontal line represents the reference line of
zero difference. P(time) value is for the change of difference over measurement
points.
77
4.7
Intravital fluorescence microscopy
In Study I, arterial diameters decreased and venous diameters increased in both
study groups. Arterial circulation ceased earlier in the necrotizing pancreatitis
group. Of the pigs with necrotizing pancreatitis, 20% had continuing arterial
circulation 30 minutes after the induction of necrotizing pancreatitis. In
comparison, 75% of the pigs with oedematous pancreatitis had arterial circulation
at the 30-minute time point. The endothelial lining of the venous walls was
destroyed during the 180-minute study period in both groups.
Fig. 14. Artery (a) and vein (b) recorded by intravital microscopy 5 minutes after the
induction of necrotizing pancreatitis. Adherent leukocytes are attached to the venous
walls.
78
Fig. 15. In intravital microscopy, image artery is no longer visible after 180 minutes
following the induction of necrotizing pancreatitis. The endothelial lining of the
venous wall (b) is destroyed.
4.8
Microdialysis
In Study I, the pancreatic extracellular fluid content of protein, albumin and
potassium increased in the necrotizing pancreatitis group, while they remained at
an unchanged level in the oedematous pancreatitis group (Pt*g = 0.002; Pt*g =
0.022; and Pt*g = 0.006, respectively).
4.9
Histological severity of the pancreatitis
In Study I, necrosis and haemorrhage were seen in the head and corpus of the
pancreas in every pig with necrotizing pancreatitis, both visually and
microscopically. Caudal parts of the pancreas were spared from necrosis. Necrosis
was seen as soon as 30 minutes after the induction of necrotizing pancreatitis.
There was oedema but no necrosis or haemorrhage in any pig in the oedematous
pancreatitis group. Accumulation of lymphocytes, neutrophils and macrophages
in the pancreatic tissue was seen in both groups.
In Studies II and III, in the necrotizing pancreatitis group there was elevation
of the interlobar, interacinar and intercellular oedema in the caput of the pancreas
79
as compared to the mild pancreatitis and control groups. Leucocytes also
increased in the borders of the necrosis throughout the pancreas but only in the
necrotizing pancreatitis group. Cell necrosis, fat necrosis, leucocyte margination,
vacuolisation and haemorrhage were detected only in the necrotizing pancreatitis
group. The pancreatic histology was identical in the oedematous pancreatitis and
control groups.
4.10 Immunohistochemistry and histology
In Study I in the baseline samples, claudin-2 immunoreactivity was seen only in
ductal cells, while the acinar cells remained negative. In the samples with
pancreatitis, membranous immunoreactivity in the acinar cells appeared, while
immunoreactivity in ductal cells remained the same. The claudin-2
immunoreactivity appeared in both necrotizing and non-necrotizing pancreatitis
samples and there was no apparent difference in the extent of immunoreactivity.
For other claudins, both acinar and ductal cells showed strong immunoreactivity
for all of them. There were no apparent changes in claudin-3, -4, -5 or -7 staining,
before and after pancreatitis had been induced in either group.
In Study II, the basic histology of the colon and jejunum did not differ
between the study groups. Epithelial cell height in the jejunum, mucosal thickness
in the colon and villus height and crypt depth in the jejunum and colon remained
the same during the 6 hour study period. Furthermore, the thicknesses of the
submucosa and serosa exudate were constant in the jejunum and colon. There
were no changes in claudin-2, or -5 staining between the study groups. The
saturation and the proportional area of claudin-3 staining decreased in the
necrotizing pancreatitis group compared to the oedematous pancreatitis group in
the upper half of the mucosa in the colon (p=0.028, p=0.028). The saturation of
claudin-4 staining decreased in the villus of the jejunum in the necrotizing
pancreatitis group as compared to the oedematous pancreatitis group (p= 0.027).
The total target area of claudin-7 decreased in the villus of the jejunum in the
necrotizing pancreatitis group as compared to the oedematous pancreatitis group
(p=0.019) and the control group (p=0.048). The saturation of claudin-7 staining
decreased in the crypts of the jejunum in the necrotizing pancreatitis group as
compared to the oedematous pancreatitis group (p=0.038). In addition, the
scanned area decreased in the necrotizing pancreatitis group as compared to the
control group in the villus of the jejunum (p=0.037). The total target area of
claudin-7 staining decreased at the bottom of the mucosa in the colon in the
80
necrotizing pancreatitis group as compared to the control group (p=0.053, p-all
groups =0.061). (Table 3-4). All other measured variables concerning claudins 3,
4 and 7 did not change.
In Study IV, there were no statistically significant changes in the expression
of E-cadherin or β-catenin immune reactivity in the colon although there was
some decrease in the expression of E-cadherin immune reactivity in the
oedematous and necrotizing pancreatitis groups (p= 0.038 proportional area in the
upper half of the mucosa). In the upper half of the mucosa in the colon, there was
decrease in saturation, intensity, density x area, total target area and proportional
area of E-cadherin immune reactivity in both the oedematous and necrotizing
pancreatitis groups. In the lower half of the mucosa, the saturation and intensity
of the E-cadherin immune reactivity decreased only in the necrotizing pancreatitis
group.
Fig. 16. Claudin-2 staining is expressed only in ductal cells before pancreatitis is
induced.
81
Fig. 17. Claudin-2 staining is expressed in the intercellular spaces and in the
cytoplasm and cell membranes of acinar cells after the induction of pancreatitis.
Ductal staining remains.
4.11 Apoptosis
In Study I, apoptosis increased in the pancreas in the oedematous pancreatitis
group whereas it remained constant in the necrotizing pancreatitis group
(oedematous pancreatitis P = 0.025; necrotizing pancreatitis P = 0.41).
In Study II, apoptosis increased in the colon in all study groups but not in the
jejunum in the necrotizing pancreatitis group. There was no difference between
the groups.
4.12 Cell proliferation
In Study II, there was no significant change in Ki-67 activity between the study
groups allthough there was a slight increase in Ki-67 activity in the colon and
jejunum in the necrotizing and oedematous pancreatitis groups. The Ki-67 activity
was noticed to have increased in the upper half of the villi.
82
4.13 Portal vein blood cultures and mesenteric lymph node bacterial
cultures
In Study II, portal vein blood cultures and mesenteric lymph node bacterial
cultures remained negative in both necrotizing pancreatitis and control groups.
One oedematous pancreatitis pig had 4x102 bacteria in venous blood at 360 min
and 2.2x 102 bacteria in the lymph node at 120 min.
4.14 Transmission electron microscopy
In Study IV, in the necrotizing pancreatitis group, the height and density of the
glycokalyx and microvilli in the colonic epithelial cells decreased. AJs and TJs
were occasionally open in the upper half of the colonic mucosa. The mitochondria
of the mucosal epithelial cells were degenerated and disrupted in the necrotizing
pancreatitis group. The endothelial cells were degenerated and the arteries were
constricted in the jejunal mesentery. Endothelial cells were vacuolated, indicating
degeneration in the mesenteric vein. Mesenteric capillaries were ruptured and the
lumens were narrowed. The capillary endothelial linings were broken and the
lumens of the capillaries were narrow in both upper and lower halves of the
mucosa. Connective tissue belts were seen around the mucosal capillaries. In the
jejunum, the intestinal microvilli remained intact in all groups. In the necrotizing
pancreatitis group, the AJs and TJs were seldom open and the lumens of the
capillaries were commonly narrowed.
The height and density of the glycokalyx in the colon were decreased in the
oedematous pancreatitis and control groups but there were no alterations in the
colonic epithelial cells´ microvilli. The mitochondria of the colonic mucosal
epithelial cells were swollen in the oedematous pancreatitis group but remained
intact in the control cases. AJs and TJs remained intact. In the oedematous
pancreatitis group, the endothelial cells of the mesenteric artery were flattened.
Endothelial cells remained intact in the control case. Endothelial cells in the
mesenteric veins were intact in both oedematous pancreatitis and control groups.
Mesenteric and mucosal capillaries were intact and open in both oedematous
pancreatitis and control groups.
83
84
540 min. Tight (o) and adherens (*) junctions are occasionally open.
) is poorly developed. c. Necrotizing pancreatitis after
) are normal. TJs and AJs junctions are intact. b. Necrotizing pancreatitis after 540
min. The height and amount of microvilli (→) are decreased. Glycocalyc (
a. Baseline. Microvilli (→) and glycocalyx (
after 540 mins follow up.
Fig. 18. Transmission electron micrograph analysis of the epithelium of colon before the induction of necrotizing pancreatitis and
Fig. 19. Transmission electron micrograph analysis of mitochondria in the colon
mucosa before and after 540 mins follow up in a control case and oedematous and
necrotizing pancreatitis.
a. Baseline. Mitochondria (*) are intact. b. Control case after 540 min. Mitochondria (*)
are intact. c. Oedematous pancreatitis after 540 min. Mitochondria (*) are swollen. d.
Necrotizing pancreatitis after 540 min. Mitochondrial (*) membranes and cristae are
disrupted and degenerated.
85
Fig. 20. Transmission electron micrograph analysis of artery and vein in jejunal
mesentery before the induction of necrotizing pancreatitis and after 540 mins follow
up.
a. Artery at baseline. Normal endothelial cells (*) and smooth muscle cells (→). b.
Artery at 540 min in necrotizing pancreatitis. The endothelial cells (*) are degenerated
and the smooth muscle cells (→) are constricted. c. Vein at baseline. Normal
endothelial cells (*). d. Vein at 540 min in necrotizing pancreatitis. Endothelial cells (*)
are vacuolated indicating degeneration.
86
5
Discussion
5.1
The porcine model
In this prospective, randomized, controlled porcine model, we were able to
reliably generate severe necrotizing pancreatitis and mild oedematous
pancreatitis. The porcine model was chosen due to the research groups´ vast
previous experience of using pigs as experimental animals. The invasive
cannulations, continuous surveillance of central haemodynamical variables and
repeated sampling of femoral arterial, portal and central venous blood used in this
model required an animal with sufficient size, such as a pig. The animal size also
allowed simultaneous use of microdialysis and intravital microscopy. The disease
course is also more rapid with rodents than with pigs, where it resembles more
closely the human disease course.
The porcine pancreatitis model has been previously documented and it
facilitated a simultaneous comparison of oedematous and necrotizing pancreatitis.
The taurocholate duct injection model is one of the best experimental pancreatitis
models. It has clinical relevance, since it has a similarity to human biliary
pancreatitis. The taurocholate duct injection model also induces multiple organ
failure, similar to that in the human disease, and is thus ideal for studying such
things as the effects of acute pancreatitis in the intestine, as we did in this study
(Ruud et al. 1982, Kinnala et al. 1999, Kinnala et al. 2002)
Caerulein or CDE-diet induced pancreatitis techniques are widely applicable
with rodents but these models are clinically less relevant, since these aetiologies
don´t occur in the human disease. CDE-diet, immunological and hypovolemia
induced models themselves cause organ damage to other organs as well and thus
would not have been suitable for studying the intestinal manifestations of
pancreatitis as we did in these experiments. Caerulein and closed duodenal loopmodels also increase bacterial translocation and thus wouldn´t have been suitable
in this study. The closed duodenal loop- model would have caused greater
surgical trauma and the onset of the disease is, in this case, too slow and the
disturbance of pancreatic microcirculation results in low repeatability.
Porcine anatomy and physiology bears a closer resemblance to humans’ than
that of mice or rats. The gastrointestinal anatomy of a pig has differences
compared to that of humans but the physiology is similar. A pig is omnivorous, as
are humans, contrary to rodents. Pigs are also monogastric, as are humans. The
87
long small intestine is mainly located in the right side of the abdomen. The
mesenteric vessels form the arcade in subserosa and not in the mesentery as
humans. Mesenteric lymph nodes in pigs are prominent. The spiral colon consists
of caecum, ascending, transverse and descending colon and it is located in left
upper quadrant of the abdomen. Sigmoid colon and appendix are absent. The liver
contains six lobes and a gall bladder (Swindle & Smith 1998). The common bile
duct and pancreatic duct enter the duodenum separately but the structure of the
sphincter of Oddi is similar to that of humans´ (Sand et al. 1993, Sand et al.
1994). Pigs´ pancreas is sizeable. Functionally, the liver and pancreas are alike to
humans’ and thus the swine has been the primary species of interest in
xerographic processes (Swindle & Smith 1998). The intestinal flora differs
between animal species (van Minnen et al. 2007)
20% taurocholic acid was used to ensure that all animals developed the
severe disease during the surveillance time of 180 to 540 minutes, even though
15% taurocholic acid has been reported to induce necrotizing pancreatitis in pigs
(Kinnala et al. 1999).
Altogether, 22 animals were used as pilots to standardize the experimental
protocol, optimize the model in regards of fluid management and optimize the
utilization of the measuring methods used. By doing these pilots, we were able to
minimize the effect of learning curve to the results. Only 3 animals were excluded
from the analysis due to technical complications. All animals included in the
studies survived the whole follow-up time.
Necrotizing pancreatitis was characterized by necrosis visible to the eye 30
minutes after the intraductal injection of taurocholic acid. The intraductal
injection of taurocholic acid mimics the etiology of biliary pancreatitis. In
humans, however, the necrosis develops during 24 to 48 hours (Balthazar 2002).
The histological changes in necrotizing pancreatitis included elevation of
oedema in the caput of the pancreas, increase of leucocytes in the borders of the
necrosis, cell and fat necrosis, leukocyte margination, vacuolisation and
haemorrhage. Blood lipase and amylase levels also rose significantly. Pancreatic
histology was identical in both oedematous pancreatitis and control groups but
elevated blood lipase levels were associated with mild oedematous pancreatitis.
5.2
Limitations of the study
All studies were done with the experimental porcine model and in spite of the fact
that the pigs´ anatomy and physiology resembles that of humans’, caution must be
88
taken when applying the results to humans. All findings should be verified in a
study with human patients.
With rodents, it is possible to perform experiments using at least two
dissimilar experimental models. This is beneficial in reducing the effect of modelspecific responses to the results. This was not possible when using pigs as the
experimental animal. However, since the taurocholate duct injection model is one
of the best models, adding an inferior model (e.g. closed duodenal loop or
ischemia induced- model) which would have been suited for use in large animals,
wouldn´t have reduced the model specific responses but increased them.
The first study lacked a control group which presents a risk of the
experimental procedure itself influencing the results. However, our finding of
increased apoptosis in oedematous pancreatitis is in line with previous findings
(Bhatia 2004b). The increase of pancreatic claudin-2 expression is not likely to
originate from the experimental procedure itself, since we demonstrated in Study
II that claudin-2 expression didn´t increase in either the jejunum or the colon.
Control groups were used in Studies II to IV.
The bacterial translocation to mesenteric lymph nodes or portal vein blood
did not occur during the 360 minutes surveillance time. A longer surveillance time
or more sensitive measuring techniques would have been required to demonstrate
whether or not the bacterial translocation occurs in porcine pancreatitis. The
evidence of early bacterial translocation is from studies with mice and rats and
thus it is possible that it occurs in a later phase of the disease in pigs.
The longest surveillance time was 540 minutes in our series. A significantly
longer follow-up time would have required changing the study protocol from the
acute model to necessitating waking up the animals from the anaesthesia. This
would have been challenging in regards of treating the pain in an ethical way.
Also, we would then have observed mortality in at least the necrotizing
pancreatitis group.
No statistical significant differences were observed in Study III in the portal
vein cytokine levels between the study groups, perhaps owing to the small sample
size. The fact that the quantity of animals was decided without a power analysis is
a limitation and may have affected the results, since the differences between
groups were small. Increasing the sample size might have produced more reliable
results but the number of animals is necessarily limited in an expensive, large
animal model.
Since our aim was to investigate the early pathogenesis of acute pancreatitis,
we chose to measure the cytokines from portal venous blood. Cytokines released
89
from the pancreas may also enter the lymphatic system in addition to the portal
vein. On the other hand, the portal vein blood also contains cytokines released
from the gut. In the future, measuring the cytokine levels from gut lymph would
serve in achieving a fuller picture of the pathogenesis of AP.
The expression of TJ and AJ proteins in Studies I, II and IV were analysed by
immunohistological staining with quantitative measuring by a blinded
investigator. Additional, precise quantitative methods would have increased the
credibility of the results.
5.3
Blood cells
In acute pancreatitis in rats, neutrophils and leukocytes were attracted to the site
of injury as early as 3 hours after the disease onset (Shanmugam & Bhatia 2010).
However, before extravasation, the leukocytes are activated and start rolling along
activated endothelial cells before forming adhesions and transmigrating through
the endothelium (Ebnet 1999). Considering this time-line, the surveillance time of
360 minutes was applied.
Activation of both innate (granulocytes, especially neutrophils) and adaptive
(mononuclear cells, macrophage, T- and B-lymphocytes) immune systems is
involved in the systemic inflammatory response in acute pancreatitis (Beger et al.
2000). In acute pancreatitis, infiltrating polymorphonuclear neutrophils produce
oxygen-free radicals and thus cause local and systemic complications (Beger et
al. 2000). In this study, the amount of neutrophils increased in all groups, perhaps
due to SIRS caused by the experimental procedure itself.
The monocyte count increased in both oedematous and necrotizing
pancreatitis in Study I but only in necrotizing pancreatitis in Study III. Monocytes
migrate to the pancreatic interstitium from the circulation. Monocytes and
macrophages produce cytokines and other inflammatory mediators (Shrivastava
& Bhatia 2010). The degree of monocyte activity has also been related to disease
severity, as our results also imply. The degree of monocyte activity contributes to
the cell death pathways of the pancreatic cells and thus the degree of local
damage (Liang et al. 2008).
During the early phase of acute pancreatitis, the lymphocytes also infiltrate
the pancreas (Shanmugam & Bhatia 2010). Systemic inflammation in acute
pancreatitis also correlates with the activation of Th-lymphocytes. Th1-cells
produce pro-inflammatory cytokines and Th 2-lymphocytes secrete antiinflammatory cytokines (Lichtman & Abbas 1997). In this study, the amounts of
90
lymphocytes decreased in all groups due to SIRS caused by the experimental
procedure itself.
During acute pancreatitis, platelets are activated and accumulate intra- and
extravascularly forming microthrombi. It was postulated that patients with
pancreatitis of differing levels of disease severity and experimental severe acute
pancreatitis were connected by a significant decrease in platelet count. In mild
human pancreatitis, platelets were activated but the amount did not decrease due
to a fast bone marrow response (Kakafika et al. 2007, Mimidis et al. 2004). Our
results showed a decreased platelet count in both necrotizing and oedematous
pancreatitis in pigs.
According to this study, the eosinophil count decreased in all groups but the
decrease was more rapid in the pancreatitis groups. It has been observed that
eosinopenia is a part of the normal response to stress and inflammation (Wardlaw
1994). It remains to be elucidated whether the degree of eosinopenia would
correlate with the disease severity in pancreatitis.
5.4
Portal vein cytokines
The involvement of many of the cytokines occurs early in the course of acute
pancreatitis and is thus impossible to study in human patients, perhaps excluding
post-ERCP pancreatitis patients. Our experimental pancreatitis model provides an
opportunity to investigate cytokine function early in the course of the disease. We
investigated several cytokines, which had not as yet been studied in experimental
acute pancreatitis. TNF-α, IL-1β, IL-10 have been shown to rise after 6 to 12
hours of severe acute pancretitis in pigs (Yekebas et al. 2002, Yang et al. 2004,
Yan et al. 2006, Li et al. 2007, Tao et al. 2008). Thus the 360 minutes
surveillance time was chosen.
In our study, PDGF increased only in necrotizing pancreatitis. The role of
PDGF has not yet been established, either in experimental pancreatitis or in the
human disease. PDGF acts as an inflammatory mediator, recruting monocytes and
neutrophils (Deuel et al. 1982). The increase in PDGF concentration in our results
may explain the increased monocyte count in necrotizing pancreatitis. PDGF is
also released from platelets during blood clotting and blood vessel endothelia at
sites of injury. PDGF promotes wound healing since it is a potent mitogen of
fibroblasts, glial cells and smooth muscle cells (Deuel & Huang 1984) and it has
been shown to promote the structural integrity of the vessel wall (Lindahl et al.
91
1997). Further studies are needed to confirm whether or not PDGF is associated
with the development of a more severe pancreatitis form.
Eotaxin has not been studied in acute pancreatitis before. At first, it was
discovered to have eosinophil chemoattractant activity (Conroy et al. 1997). Since
then, it has been associated with pro-inflammatory effects in early acute sepsis
(Osuchowski et al. 2006) and, in combination with other cytokines it may have
some prognostic value in predicting mortality in sepsis (Punyadeera et al. 2010).
According to our results, eotaxin increased both in oedematous and necrotizing
pancreatitis and might have an effect in the pathogenesis of acute pancreatitis.
MCP-1 is secreted by monocytes, fibroblasts and vascular endothelial cells
and it is known to attract monocytes, T lymphocytes, natural killer cells and
neutrophils to the inflammation site (Robertson & Coopersmith 2006, Marra
2005). MCP-1 was previously shown to correlate with the incidence of organ
damage in acute pancreatitis (Papachristou et al. 2005, Rau et al. 2003). In our
results, MCP-1 increased markedly in all groups indicating that it also has an
effect on mediating systemic response to laparotomy itself.
IL-6 and IL-8 have been shown to possess early prognostic value in
determining disease severity in acute pancreatitis (Aoun et al. 2009). IL-6 has
both pro- and anti-inflammatory effects. It stimulates the synthesis of acute phase
proteins in the liver (Castell et al. 1989) and, on the other hand, it prevents the
synthesis of pro-inflammatory cytokines IL-1β and TNF-α (Opal & DePalo
2000). IL-8 is synthesized by, for example, monocytes, endothelial cells and
neutrophils. It is a pro-inflammatory chemokine that plays a role in neutrophil
chemoattraction, degranulation and release of elastase (Gross et al. 1992). Our
results have shown that there was a slight increase in IL-6 only in necrotizing
pancreatitis after 120 minutes. IL-8 peaked after 120 minutes and decreased
thereafter in both oedematous and necrotizing pancreatitis
IL-9, IL-4, MIP-1α and IFN- γ concentrations did not change in our results.
IL-9 has not been studied in acute pancreatitis before. A longer surveillance time
would have been required to study whether or not these cytokines are influential
in the pathogenesis of porcine experimental pancreatitis.
Previous studies have shown that concentrations of anti-inflammatory IL-4
were low in human patients with acute pancreatitis 1 to 21 days after hospital
admission and, in patients with post-ERCP pancreatitis, 24 hours after ERCP
(Mentula et al. 2004, Kilciler et al. 2008). Severe acute pancreatitis patients with
infection had higher concentrations of IL-4 on days 7 and 14 after the hospital
admission than SAP patients without infection (Shen et al. 2011).
92
In human pancreatitis patients, macrophage inflammatory protein-1alpha
(MIP-1α) levels remained unaffected by local complications and showed a
significant increase only if MODS developed or patients subsequently died (Rau
et al. 2003).
IFN- γ was found at high concentration levels in human AP on hospital
admission (Bhatnagar et al. 2003) and SAP within 72 hours after hospital
admission (Gong et al. 2010). During the early stage of human AP, the
concentration of IFN- γ increased more in the severe cases compared with those
who had milder symptoms (Uehara et al. 2003). IFN-gamma can have antiinflammatory effects on acute pancreatitis by depressing the proinflammatory
consequences of NF-kappaB activation (Hayashi et al. 2007, Rau et al. 2006).
Cytokines and chemokines form a complex network that is activated during
critical illness. The interactions are still poorly understood (Bellomo 1992).
Cytokines are pleiotrophic, acting on different cell types causing different effects,
and the same effect can be induced by different cytokines (Commins et al. 2010).
A dynamic balance exists between pro- and anti-inflammatory reactions. Acute
phase response is controlled by a decrease in pro-inflammatory mediators and an
increase in endogenous antagonists. However, when the balance is not
accomplished, a massive systemic reaction launches and the effect of the
cytokines becomes deleterious ( Davies & Hagen 1997).
The therapeutic window of opportunity is narrow for anti-inflammatory
treatments since AP patients often present with organ failure already present on
hospital admission. Patients with CARS and immune suppression could benefit
from immunostimulation. However, the peripheral blood could show
immunosuppression and other organs might still be in the pro-inflammatory stage
(Kylanpaa et al. 2010). Thus, it would be of importance to be able to recognize at
what stage of the disease the patient is at. A better understanding of the cytokine
pattern involvement in acute pancreatitis may enable doctors to forecast the
disease severity and provide targeted immune-modulation therapy.
Due to complexity of the cytokine pattern, it is likely that measuring a group
of cytokines is more beneficial in monitoring the stage of the disease than
measuring a single cytokine. The multiplex method used in our study enables the
monitoring of the cytokine pattern. This is an advantage compared to the most
widely used ELISA method, in which only a single protein can be measured in
each sample. Multiplex technology is promising but it is not yet the golden
standard of cytokine measurement due to limited experience (Leng et al. 2008).
93
5.5
Intravital fluorescence microscopy (IVM)
In previous studies using IVM, the first signs of microcirculatory vasoconstriction
were visible just 2 minutes after the onset of pancreatitis, and leukocyte
adherence just 6 minutes after, in rats (Kusterer et al. 1993). Thus, the follow-up
time of 180 minutes was applied.
Intravital fluorescence microscopy showed that arterial circulation diminished
in both oedematous and necrotizing pancreatitis. The decrease was more
prominent in necrotizing pancreatitis. This is in line with previous findings of
severe pancreatitis being associated with reduced tissue perfusion (Liu et al.
1996, Bassi et al. 1994, Kusterer et al. 1993, Plusczyk et al. 2003) but differs
from the finding that mild pancreatitis is associated with relative hyperaemia,
increased pancreatic oxygen tension and increased oxygen saturation of
haemoglobin (Kinnala et al. 2001, Foitzik et al. 1994a). Venous dilatation was
seen in both groups in our IVM- results.
IVM offers a method of monitoring leukocyte to endothelial cell interaction
and perfusion failure in-vivo. However, in this study, it did not perform optimally
because of the early cessation of the pancreatic head microcirculation.
5.6
Microdialysis
In Study I, the increase in the pancreatic extracellular fluid content of protein,
albumin and potassium in necrotizing pancreatitis is most likely due to increased
vascular permeability but also due to pancreatic cell and pancreatic vascular wall
necrosis. It would have been of interest to study the vascular permeability from
other organs as well and to monitor the general vascular permeability disorder
related to SIRS.
Several attempts had been made to analyze, for example, different cytokines
from pancreatic extracellular fluid using the ELISA method. These analyses failed
because unfortunately the multiplex method was not yet available then.
5.7
Immunohistochemistry and histology
Claudin-5 (CL-5) immunostaining has been shown to decrease in the pancreas 3
to 12 hours after severe pancreatitis in rats (Xia et al. 2012). Disruption of
occludin and claudin-1 and up-regulation of claudin-2 were observed in the ileal
mucosa of rats 6 hours after the onset of pancreatitis (Lutgendorff et al. 2009).
94
Since there are no previous porcine studies concerning claudin expression in
acute pancreatitis, the surveillance time of 180 minutes was applied in Study I, to
analyze claudin expression in the pancreas, and 360 minutes in Study II, to
analyze claudin expression in the jejunum and colon.
CL-2 immunoreactivity in the pancreas was increased in both oedematous
and necrotizing pancreatitis. In the baseline samples, CL-2 immunoreactivity was
seen only in ductal cells. During acute pancreatitis, CL-2 immunoreactivity
appeared in the membranes of acinar cells but remained unchanged in ductal
cells. CL-2 has been found to increase the leaking of a monolayer (Anderson et
al. 2009) and thus it may contribute to oedema formation and leukocyte
infiltration of pancreatic tissue in acute pancreatitis.
Immunoreactivity of CL-3, -4, -5 and -7 remained the same in the pancreas in
both oedematous and necrotizing pancreatitis, suggesting that they do not have a
role in the development of pancreatic oedema.
In rats´ ilea, villous height was decreased eighteen hours after the disease
onset (Nakajima et al. 2007). 24 hours after the disease onset, broadened villi and
inflammatory cell infiltration were seen in rats’ ilea (Mikami et al. 2009). 8 hours
after onset, intestinal cell swelling, inflammatory cell infiltration, congestion and
lamellar haemorrhages were seen in the rats’ colons (Wang et al. 2009). The
villous height, villous height/- crypt depth and mast cell index were significantly
reduced in three pancreatitis patients during the late phase of the disease (Ammori
et al. 2002). There is no previous data about the intestinal histological changes in
acute porcine pancreatitis. Our results indicate that there were no changes in
epithelial cell height in the jejunum, mucosal thickness in the colon, or villus
height and crypt depth in either the jejunum or colon 6 hours after the disease
onset. Also, the thicknesses of the submucosa and serosa exudate were constant in
the jejunum and colon.
Previously, only a few TJ and AJ junction proteins have been analyzed from
the intestine in acute pancreatitis. These include claudin-1, -2 and occludin.
Claudin-1 decreased and occludin disrupted the ileal mucosa of rats early in acute
pancreatitis (Lutgendorff et al. 2009). In this study, we analyzed claudins -2, -3, 4, -5 and -7.
CL-2 and -5 expressions remained at the same level in the jejunum and colon
in both oedematous and necrotizing pancreatitis. The CL-2 result is the reverse of
previous results found in rats. Claudin-2 has been shown to be up-regulated in the
ileal mucosa of rats 6 hours after the onset of the disease (Lutgendorff et al.
2009). However, the disease course might be faster in smaller animals and a
95
longer surveillance time would have been required to monitor whether or not CL2 expression is affected in porcine pancreatitis.
The saturation and the proportional area of the claudin -3 staining decreased
in the upper half of the mucosa in necrotizing pancreatitis. In the jejunum, the
saturation of claudin -4 staining decreased in the villi as well as the saturation in
claudin -7 staining in the cryptae of necrotizing pancreatitis. These results
indicate that claudins mostly seem to remain unaltered during the early phase of
the disease but claudins -3, -4 and -7 might, in some proportion, decrease in acute
necrotizing pancreatitis, which may lead to tight junction disruption and bacterial
translocation during the later course of the disease.
Our electron microscopy data suggests that 540 minutes after the onset of the
acute necrotizing porcine pancreatitis, the mucosal integrity of the colon might be
affected by the opening of some of the mucosal TJs and AJs. There was some
decrease in the expression of E-cadherin immune reactivity in both oedematous
and necrotizing pancreatitis groups in the colon although the change was not
statistically significant. The decrease of E-cadherin could affect the integrity of
AJs.
Multiple mechanisms not analyzed in this study affect intestinal integrity.
Also, claudin function is regulated by several mechanisms which we did not
analyze; for example, endocytic recycling and claudin internalization were not
measured in this study (Matsuda et al. 2004, Utech et al. 2010, Findley & Koval
2009).
5.8
Apoptosis
In previous studies with rats, apoptosis was shown to occur in the small intestine
6 hours after the onset of pancreatitis (Zhang et al. 2007b). This is why we
monitored the animals for 360 minutes in this study.
In Study I, apoptosis increased in the pancreas of oedematous pancreatitis
which is in line with previous reports. Apoptosis in the pancreas is beneficial to
the disease course and has been associated with the milder disease form (Bhatia
M 1998).
In Study II, apoptosis increased in the colon of all study groups but not in the
jejunum in necrotizing pancreatitis. Our result suggests that apoptosis was
induced by the experimental procedure itself although it has been reported that in
acute pancreatitis apoptosis also occurs in the small intestine and the degree of
apoptosis correlates with the severity of mucosa barrier decline (Zhang et al.
96
2007b) . It would be of interest to study the apoptosis rate in the intestine in the
later phase of the disease in porcine experimental pancreatitis.
5.9
Cell proliferation
Cell proliferation is the measurement of the number of cells that are dividing. The
intestine is a continually renewing tissue. Its cellular turnover allows its entire
epithelium to be replaced every 3 to 5 days (Cheng & Leblond 1974). Under
normal conditions, homeostasis is maintained with cell proliferation and cell loss
by apoptosis or exfoliation into the lumen. Cell proliferation has not been
measured in the intestine in acute pancreatitis before. Since cell proliferation and
apoptosis are simultaneous processes in normal conditions, the same surveillance
time was applied in our pancreatitis model as for monitoring apoptosis.
Cell proliferation occurs in proliferative compartments near the base of the
crypt. Cells migrate and differentiate from there onto the villi (Yen & Wright
2006). Apoptotic bodies are found towards the distal end of the known cellular
migration routes. The adjacent epithelial cells and sub-epithelial macrophages
remove the apoptotic bodies by engulfment. The clearance of the apoptotic bodies
is rapid, lasting only 1 to 2 hours (Hall et al. 1994).
In Study II, there was an increase in Ki-67 activity, in particular in the upper
half of the mucosa in the colon and villi in the jejunum in both oedematous and
nectizing pancreatitis. This could be a protective mechanism to maintain cell
numbers in the villi during acute pancreatitis. We didn´t measure the cell loss by
exfoliation and we can´t tell whether it increased or not. Apoptosis increased in all
groups. Increased cell proliferation in the upper half of the mucosa and villi might
also reflect increased cell turnover since, under normal conditions, cell
proliferation occurs in proliferative compartments in the cryptae.
5.10 Portal vein blood cultures and mesenteric lymph node bacterial
cultures
In Study II, bacterial translocation to the mesenteric lymph nodes or portal vein
blood did not occur during the 360 minutes surveillance time. A longer
surveillance time or a more sensitive measuring technique such as, for example,
detecting bacterial DNA using the polymerase chain reaction method, would have
been required to demonstrate whether or not bacterial translocation occurs in
porcine pancreatitis. In studies with rats, it has been shown that bacterial
97
translocation has already occured 180 to 360 minutes after the induction of
pancreatitis and thus the surveillance time of 360 minutes was deployed (Samel et
al. 2002, Lutgendorff et al. 2009). Those studies used intravital microscopy and
fluorimeter measurement, which are more sensitive measures than the bacterial
cultures used in our study. It is possible that the bacterial translocation occurs in a
later phase of the disease in pigs.
5.11 Transmission electron microscopy
Previous studies using electron microscopy to analyze intestinal ultrastructure in
pancreatitis have been done with rats and dogs. In necrotizing pancreatitis in rats,
3 hours after the disease onset, the mucosa was not intact and inflammatory cells
were seen infiltrating the propria, submucous and serosal layers (Zhang et al.
2009b). In rats, 24 hours after the disease onset, the microvilli of the iliac mucosa
were shortened and at some points had disappeared (Wang et al. 2001a, b). 18 hours
after the onset of pancreatitis in dogs, there was a marked destruction of villi and
widening of the intervillous space in the terminal ileum (Takagi & Isaji 2000). As
the first changes were seen after 3 hours in rats’ intestines, a surveillance time of 540
minutes was applied in this study.
According to our results, 540 minutes after the onset of acute porcine
pancreatitis, damage in glycocalyx and microvilli are present in the colon. These
changes are more prominent in necrotizing pancreatitis. In both pancreatitis
groups, the microvilli remain unaltered in the jejunum.
Our observations indicate that the height and density of the glycocalyx are
decreased in both oedematous and necrotizing pancreatitis, but these changes
were clearly more advanced in necrotizing pancreatitis. Abnormalities observed in
the clygocalyx in the present study likely provide a partial explanation for the
epithelial dysfunction. Further studies are needed to see whether abnormalities in
glyxocalyx are the result of a general dysfunction of the epithelial cells, or
whether there is a specific aberration of metabolism, such as glycosylation of
individual proteins of the glygocalyx.
According to our findings, TJs and AJs were occasionally open in the
epithelium of the colon and seldom open in the epithelium of the jejunum in
necrotizing pancreatitis. In oedematous pancreatitis, they were intact in both
colon and jejunum. Previous observations in rats have indicated that tight and
intermediate junctions of the epithelial cells of the ileac villi in necrotizing
pancreatitis showed breakage 24 hours after the onset of the disease (Liu et al.
98
2009). On the other hand, contradictory evidence exists: in another study, no
abnormal changes in the junctional complex of epithelial cells of rats were found
24 hours after the onset of the disease (Wang et al. 2001b).
The finding of occasionally open AJs in both colon and jejunum in
necrotizing pancreatitis leads us to question whether there would be changes in Ecadherin and β-catenin expression which would possibly explain the AJs being
open. AJs are formed by interactions between transmembrane proteins such as Ecadherin which are connected to the cytoskeleton by intracellular proteins such as
catenins (Groschwitz & Hogan 2009b). E-cadherin-catenin complexes link cells
together, help maintain cell polarity and regulate epithelial migration and
proliferation (Perez-Moreno et al. 2003, Ebnet 2008, Reynolds & RoczniakFerguson 2004). We observed a decrease in the expression of E-cadherin immune
reactivity in the colon in both oedematous and necrotizing pancreatitis. However,
the differences were not statistically significant, probably owing to the small
sample size (n=8 per group). There are no previous studies concerning E-cadherin
function in the intestine in acute pancreatitis.
In our results endothelial cell degeneration was present in necrotizing
pancreatitis. It was seen in mesenterial arteries and veins and also in mucosal
capillaries. In necrotizing pancreatitis, mucosal capillaries in the colon showed
ruptured endothelium, narrowed lumen and were encircled by collagen fibrils. In
oedematous pancreatitis, there was only minor flattening of endothelial cells in
the mesenterial arteries. These morphological findings are in line with previous
functional studies showing reduced microcirculatory blood flow in the colon in
severe AP and maintained capillary blood flow in mild experimental acute
pancreatitis (Foitzik et al. 2002, Hotz et al. 1998). However, the mechanisms of
endothelial damage remain speculative.
It has been reported that mitochondrial dysfunction plays an important role in
the progression of organ failure in critical illness (Dare et al. 2009). Significant
mitochondrial dysfunction has been found in the jejunum in the early phase of
both mild and severe acute pancreatitis in rats (Mittal et al. 2011). Morphological
changes have been shown to occur in mitochondrial structures in acute
experimental pancreatitis. In the colons of rats with necrotizing pancreatitis,
mitochondria have been shown to be swollen (Wang et al. 2001b). In our results,
the mitochondria of the epithelium of colonic mucosa were swollen in
oedematous pancreatitis and degenerated and disrupted in necrotizing
pancreatitis. Previous reports on multiorgan failure are indicative of a
compromised cellular energy state and defective mitochondrial function.
99
However, it has been found that this is not caused by faulty signaling on the part
of enzymes or of organelle biosynthesis. On the contrary, in sepsis-induced
multiorgan failure, synthesis seems to be activated but cannot compensate for
organelle destruction (Fredriksson et al. 2008)
In conclusion, in the early phase of porcine necrotizing pancreatitis, the
endothelial cell damage becomes apparent in mesenteric arteries, veins and in
colonic mucosal capillaries. This may lead to decreased mucosal tissue perfusion,
explaining the mucosal mitochondrial damage and epithelial injury in the colon in
necrotizing pancreatitis. The first signs of the beginning of gut barrier disruption
emerge as some epithelial TJs and AJs open. Acute pancreatitis may be
characterized by a decrease of E-cadherin in the colon and this may contribute to
the AJs opening. These events seem to occur very early during the disease course
in acute necrotizing pancreatitis but not in oedematous pancreatitis.
100
6
1.
2.
3.
4.
Conclusions
Claudin-2 expression increases in the membranes of acinar cells during acute
pancreatitis and its role to paracellular permeability needs further study. The
expressions of claudins -3, -4, -5 and -7 in the pancreas are unaffected during
the early stages of acute pancreatitis.
Bacterial translocation from the gut is not present at the beginning of acute
porcine pancreatitis. The expressions of claudins-2 and -5 do not become
altered. There might be some decrease in claudin-3 expression in the colon
and reduced expression of claudins-4 and -7 in the jejunum in necrotizing
pancreatitis. Laparotomy itself causes increased apoptosis in the colon and
jejunum.
The initial inflammatory process is diverse in oedematous and necrotizing
pancreatitis. Increased monocyte count in combination with elevated PDGF
and IL-6 are characteristic of necrotizing pancreatitis in our model.
Necrotizing pancreatitis causes damage to the epithelial and endothelial cells
in the colon during the early stages of the disease. The expression of Ecadherin immunoreactivity showed a trend for decrease in the colon in both
oedematous and necrotizing pancreatitis.
101
102
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128
Original publications
I
Meriläinen S, Mäkelä J, Anttila V, Koivukangas V, Kaakinen H, Niemelä E, Ohtonen
P, Risteli J, Karttunen T, Soini Y & Juvonen T (2008) Acute edematous and
necrotizing pancreatitis in a porcine model. Scand J Gastroenterol 43(10): 1259–68
II Meriläinen S, Mäkelä J, Koivukangas V, Jensen HA, Rimpiläinen E, Yannopoulos F,
Mäkelä T, Alestalo K, Vakkala M, Koskenkari J, Ohtonen P, Koskela M, Lehenkari P,
Karttunen T & Juvonen T (2012) Intestinal bacterial translocation and tight junction
structure in acute porcine pancreatitis. Hepatogastroenterology 59(114): 599–606
III Meriläinen S, Mäkelä J, Jensen HA, Dahlbacka S, Lehtonen S, Karhu T, Herzig KH,
Kröger M, Koivukangas V, Koskenkari J, Ohtonen P, Karttunen T, Lehenkari P&
Juvonen T (2012) Portal vein cytokines in the early phase of acute experimental
oedematous and necrotizing porcine pancreatitis. Scand J Gastroenterol. 47(11):
1375–85
IV Meriläinen S, Mäkelä J, Sormunen R, Jensen HA, Rimpiläinen R, Vakkala M,
Rimpiläinen J, Ohtonen P, Koskenkari J, Koivukangas V, Karttunen T, Lehenkari P,
Hassinen I & Juvonen T (2012) Effect of acute pancreatitis on porcine intestine: A
morphological study. Ultrastruct Pathol. In Press
Reprinted with permission from (I+III, IV) Informa Healthcare and (II) Thieme
Publishers.
Original publications are not included in the electronic version of the dissertation.
129
130
ACTA UNIVERSITATIS OULUENSIS
SERIES D MEDICA
1180. Norppa, Anna (2012) Association between periodontal
inflammation : A study of pro- and anti-inflammatory mediators
and
systemic
1181. Karjalainen, Teemu (2012) Nitinol shape memory alloy in flexor tendon repair
1182. Saukkonen, Tuula (2012) Prediabetes and associated cardiovascular risk factors :
A prospective cohort study among middle-aged and elderly Finns
1183. Hagman, Juha (2012) Resource utilization in the treatment of open angle
glaucoma in Finland: an 11-year retrospective analysis
1184. Eskola, Pasi (2012) The search for susceptibility genes in lumbar disc
degeneration : Focus on young individuals
1185. Kaivorinne, Anna-Lotta (2012) Frontotemporal lobar degeneration in Finland :
Molecular genetics and clinical aspects
1186. Härkönen, Pirjo (2012) Elämäntyytyväisyys ja terveys : Voimavarasuuntautunut
ikääntyvien henkilöiden seurantatutkimus
1187. Laukkanen, Päivi (2012) Occurrence of high risk human papillomaviruses and
cervical cancer among fertile-aged women in Finland
1188. Junttila, Eija (2012) Cardiovascular abnormalities after non-traumatic intracranial
hemorrhage
1189. Hookana, Eeva (2012) Characteristics of victims of non-ischemic sudden cardiac
death
1190. Murugan, Subramanian (2012) Control of nephrogenesis by Wnt4 signaling :
Mechanisms of gene regulation and targeting of specific lineage cells by tissue
engineering tools
1191. Nikkilä, Jenni (2013) PALB2 and RAP80 genes in hereditary breast cancer
predisposition
1192. Määttä, Mikko (2012) Assessment of osteoporosis and fracture risk : Axial
transmission ultrasound and lifestyle-related risk factors
1193. Kaakinen, Marika (2012) Genetic and life course determinants of cardiovascular
risk factors : Structural equation modelling of complex relations
1194. Nevalainen, Mika (2012) Gene product targeting into and membrane trafficking
from the endoplasmic/sarcoplasmic reticulum in skeletal myofibers
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