Experimental study of acute pancreatitis in a porcine model, especially tight junction structure and

134 Pages·2013·4.48 MB·English

by  Meriläinen, Sanna

D OULU 2013
D 1195
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UNIVERSITY OF OULU P.O.B. 7500 FI-90014 UNIVERSITY OF OULU FINLAND
ACTA
A UNIVERSITATIS OULUENSIS
A C T A U N I V E R S I T A T I S O U L U E N S I S C
T
A
D
S E R I E S E D I T O R S MEDICA
Sanna Meriläinen
A
SCIENTIAE RERUM NATURALIUM
S EXPERIMENTAL STUDY OF
a
B Senior Assistant Jorma Arhippainen n
n
a ACUTE PANCREATITIS
HUMANIORA
M
CUniversity Lecturer Santeri Palviainen er IN A PORCINE MODEL,
i
l
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TECHNICA in
e ESPECIALLY TIGHT JUNCTION
D Professor Hannu Heusala n
MEDICA STRUCTURE AND PORTAL
E Professor Olli Vuolteenaho
VEIN CYTOKINES
SCIENTIAE RERUM SOCIALIUM
F University Lecturer Hannu Heikkinen
SCRIPTA ACADEMICA
G Director Sinikka Eskelinen
OECONOMICA
Professor Jari Juga
EDITOR IN CHIEF
Professor Olli Vuolteenaho
PUBLICATIONS EDITOR
Publications Editor Kirsti Nurkkala
UNIVERSITY OF OULU GRADUATE SCHOOL;
UNIVERSITY OF OULU,
FACULTY OF MEDICINE,
ISBN 978-952-62-0064-4 (Paperback)
INSTITUTE OF CLINICAL MEDICINE, DEPARTMENT OF SURGERY;
ISBN 978-952-62-0065-1 (PDF) INSTITUTE OF DIAGNOSTICS, DEPARTMENT OF PATHOLOGY;
ISSN 0355-3221 (Print) INSTITUTE OF BIOMEDICINE, DEPARTMENT OF ANATOMY AND CELL BIOLOGY;
ISSN 1796-2234 (Online) INSTITUTE OF BIOMEDICINE, DEPARTMENT OF PHYSIOLOGY;
OULU UNIVERSITY HOSPITAL

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
UNIVERSITY OF OULU, OULU 2013
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 toisi-
naan 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 tii-
visliitosproteiinien 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 sytokiinipi-
toisuuksia porttilaskimoverestä. Neljännessä työssä analysoitiin ohut- ja paksusuolen mikrora-
kennetta 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. Suolis-
tossa 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 vai-
keassa kokeellisessa haimatulehduksessa. Monosyyttimäärän sekä PDGF:n ja IL-6:n pitoisuuksi-
en lisääntyminen, olivat tyypillisiä vaikealle haimatulehdukselle tässä mallissa. IV työssä todet-
tiin, 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 haima-
tulehduksessa. 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 co-
operation 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 co-
operation 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
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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 grand-
children, 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 Sanna Meriläinen

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