The effects of a heterozygous SOD2-deficiency and running exercise on the knee articular cartilage in a mouse model [Elektronische Ressource] / Jan Henkel

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Publié le : samedi 1 janvier 2011
Lecture(s) : 23
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Source : D-NB.INFO/1016743041/34
Nombre de pages : 105
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Aus dem Institut für Kreislaufforschung und Sportmedizin
der Deutschen Sporthochschule Köln
Abteilung für Molekulare und Zelluläre Sportmedizin
Geschäftsführender Leiter: Universitätsprofessor Dr. med. W. Bloch



„The effects of a heterozygous SOD2-deficiency and running exercise on the
knee articular cartilage in a mouse model‟



Inaugural-Dissertation zur Erlangung der Doktorwürde
der Hohen Medizinischen Fakultät
der Universität zu Köln



vorgelegt von
Jan Henkel
aus Marburg an der Lahn



Promoviert am 05. Oktober 2011



Dekan: Universitätsprofessor Dr. med. Dr. h.c. Th. Krieg
1. Berichterstatter: Universitätsprofessor Dr. med. W. Bloch
2. Berichterstatter: Universitätsprofessor Dr. med. P. Eysel







Erklärung

Ich erkläre hiermit, dass ich die vorliegende Arbeit ohne unzulässige Hilfe
Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt
habe; die aus fremden Quellen direkt oder indirekt übernommenen Gedanken
sind als solche kenntlich gemacht.


Bei der Auswahl und Auswertung des Materials sowie bei der Herstellung des
Manuskriptes habe ich keine Unterstützungsleistungen erhalten.


Weitere Personen waren an der geistigen Herstellung der vorliegenden Arbeit
nicht beteiligt. Insbesondere habe ich nicht die Hilfe eines Promotionsberaters in
Anspruch genommen. Dritte haben von mir weder unmittelbar noch mittelbar
geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem
Inhalt der vorgelegten Dissertation stehen.


Die Arbeit wurde von mir bisher weder im Inland noch im Ausland in gleicher
oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt.








Köln, den 30.04.2011
Jan Henkel


Die in dieser Arbeit angegebenen Experimente sind nach entsprechender Anleitung
durch Frau Dr. A. Niehoff und die medizinisch-technischen Assistentinnen Frau M.
Gilav und Frau A. Voss von mir selbst ausgeführt worden.


























ACKNOWLEDGEMENTS

I would like to address special thanks to Prof. Dr. W. Bloch and Dr. Anja
Niehoff for their guidance and insightful comments in the achievement of this
work. I would like to express my gratitude for their four years of supervision
and patience, and their commitment to guiding me through my doctoral
research. Their enthusiasm and professionalism about scientific research as well
as their support and advice have always been a valuable source of ideas and
motivation to me. The time spent discussing experiments and their results,
solving problems and finally with reading the drafts of my thesis as well as their
critical comments are greatly acknowledged. On a professional and personal
level it has been a great pleasure working with them.
I would like to thank the laboratory technicians Ms. M. Gilav and Ms. A. Voss
for all kinds of trouble shooting and for being such a great help and motivation
throughout the daily work in the laboratories.
I also would like to thank Mr. Alexander D. J. Baur with whom I had the
pleasure of working on this project. Alex has been a valuable colleague and
friend throughout the years.
I would like to thank my parents Ingelies and Wolfgang for all their support and
appreciation during all the years and for giving me a peaceful home to retreat to
whenever times were getting too stressful.
Finally, I would like to thank Prof. Dr. K. Scharffetter-Kochanek for kindly
providing the knockout mice used in this study.













‘Tho' much is taken, much abides; and though
We are not now that strength which in old days
Moved earth and heaven; that which we are, we are;
One equal temper of heroic hearts,
Made weak by time and fate, but strong in will
To strive, to seek, to find, and not to yield.’
- Alfred Lord Tennyson, Ulysses -





















Meinen Eltern Ingelies und Wolfgang
und meinen Großeltern

























































TABLE OF CONTENTS
Glossary ..................................................................................................................................... 1
I. Introduction ................................................................................................................... 2
1.1. FREE RADICALS AND REACTIVE (OXYGEN) SPECIES ............ 2
1.2. THE MITOCHONDRION AS MAIN SOURCE OF REACTIVE OXYGEN SPECIES IN THE CELL ..................... 3
1.3. CELLULAR DEFENSE SYSTEMS AGAINST REACTIVE SPECIES ............................... 4
1.4. OXIDATIVE STRESS AND OXIDATIVE DAMAGE ...................................................................................... 6
1.5. CELLULAR PROCESSES INVOLVING REACTIVE SPECIES: REDOX-HOMEOSTASIS AND SIGNALING ..... 7
1.6. GENERAL VIEW ON OXIDATIVE STRESS AND DISEASES ........................................................................ 8
1.7. MANGANESE SUPEROXIDE DISMUTASE (MNSOD, SOD2) ....... 9
1.8. PHYSICAL ACTIVITY, OXIDATIVE STRESS AND MNSOD ....... 11
1.9. OXIDATIVE STRESS, JOINT DISEASES AND SOD ................................................................................... 13
1.10. PURPOSE OF THE STUDY: CARTILAGE AND MNSOD ............ 14
II. Material and methods ................................................................................................. 15
2.1. LABORATORY ANIMALS ...................... 15
2.1.1. Genetic background and mice husbandry ...................... 15
2.1.2. Training and control groups ........................................... 15
2.1.3. Training protocol ............................................................................................ 15
2.1.4. Extraction and processing of the tissues ......................................................... 16
2.2. HISTOLOGICAL STAINING ................... 16
2.3. IMMUNOHISTOCHEMISTRY ................................................................................. 16
2.3.1. Protocol .......................................................................... 16
2.3.2. Antibodies ....................................... 17
2.3.3. Semiquantitative analysis of the immunohistochemical staining .................... 17
2.4. HISTOMORPHOMETRY: MEASUREMENT OF CARTILAGE THICKNESS ................................................ 19
2.4.1. Anti-Collagen Type ІІ-Immunohistochemistry ................................ 19
2.4.2. Measurement of cartilage thickness ............................................................... 20
2.5. STATISTICAL ANALYSIS ....................................................... 20
III. Results .......................................................................................................................... 22
3.1. PHENOTYPE, TRAINING AND BIOMETRY ............................. 22
3.2. HISTOLOGICAL STAINING PATTERN ................................... 23
3.3. IMMUNOHISTOCHEMISTRY ................................................. 40
3.3.1. Nitrotyrosine ................................................................... 40
3.3.2. 15-F -Isoprostane (8-iso-PGF ) ................................... 45 2t 2α
3.3.3. Active caspase-3 ............................................................. 50
3.3.4. Ki67 ................................................................................................................ 55
3.4 HISTOMORPHOMETRY: MEASUREMENT OF CARTILAGE THICKNESS ................................ 60
ІV. Discussion ..................................................................................................................... 63
4.1. HISTOMORPHOMETRY AND HISTOLOGICAL STAINING PATTERN ...................... 63
4.2. IMMUNOHISTOCHEMISTRY ................................................................................. 64
4.3. IMPLICATION OF THE RESULTS FOR UNDERSTANDING PATHOPHYSIOLOGY OF OSTEOARTHRITIS .. 70
V. Summary / Zusammenfassung ................................................................................... 74
5.1. SUMMARY ............................................................................ 74
5.2. ZUSAMMENFASSUNG ........................................................... 76

VI. References .................................................................................................................... 78
6.1. REFERENCES ........................................ 78
5.3. PRELIMINARY PUBLICATIONS ............. 90
V ІІ. Appendix ...................................................................................................................... 91
7.1. CHEMICALS AND DILUTIONS ............................................... 91
7.1.1. Phosphate buffer (PB) 0.1M pH 7.4 ............................... 91
7.1.2. Tris buffered saline (TBS) 0.05M pH 7.6 ........................................................ 91
7.1.3. Phosphate Buffered Saline (PBS) 0.2M [pH 7.4] ........... 91
7.1.4. 4% paraformaldehyde (PFA) in 0.1M PBS [ph 7.4] ...................................................................... 91
7.1.5. EDTA (20%): .................................................................. 92
7.1.6. Imidazole buffer [pH 7.0] (125ml) . 92
7.2. HISTOLOGICAL STAINING PROTOCOLS ............................................................... 92
7.2.1. Hematoxylin-Eosin Stain ................................................................................ 92
7.2.2. Safranin O / Fast Green Stain ........................................................................ 93
7.2.3. Masson's trichrome stain ................ 93
7.2.4. Alcian blue / nuclear fast red .......... 94
VIII. Lebenslauf .................................................................................................................... 95















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