Regulation of prostaglandin E_1tn2 release in cerebral ischemia [Elektronische Ressource] / presented by Waleed M. Adel Barakat

Wbarakat

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• Gesundheit

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2009
• Nombre de lectures: 36
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Dissertation
submitted to the

Combined Faculties for the Natural Sciences
and for Mathematics
of the Ruperto ‐Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences










presented by

Master Sci. Waleed M. Adel Barakat
born in: Belbies, El-Sharkia, Egypt
Oral ‐examination: -01-2009.





Regulation of Prostaglandin E release in cerebral ischemia 2


















Referees: Prof. Dr. Ulrich Hilgenfeldt
Prof. Dr. Markus Schwaninger




Dedicated to my family
Acknowledgements

Acknowledgements
All the experiments of my dissertation were done in the lab of Prof. Dr. Markus
Schwaninger to whom I would like to express my deepest thankfulness for giving
me the opportunity to join his lab, for his help and support both scientifically and
personally during all the duration of my stay in Germany and for providing a nice
friendly working atmosphere for me and for the whole research group, I would like
also to thank Prof. Dr. Ulrich Hilgenfeldt for his support during this dissertation.
I would like to express my deepest thanks and appreciation to my family for their
continuous love and support during my whole life which formed the perfect
environment for me to give my best effort in research.
I am also very grateful to all my colleagues and teachers at the faculty of Pharmacy,
Zagazig University, Egypt for their continuous effort and support.
This dissertation owes its existence to the help provided by many people whom I
already mentioned and others who provided equivalent support.
I am very grateful to Dr. Kevin J. Tracey (Feinstein Institute for Medical Research,
Manhasset, USA) for providing anti-HMGB1 antibody, Dr. N. Van Rooijen
(Department of Molecular Cell Biology, Amesterdam, The Netherlands) for providing
clodronate and PBS containing liposomes, Dr. B. Baumann (Institute of
Physiological Chemistry, University of Ulm, Germany) for providing the constitutively
active IKK2 constructs and mice expressing dominant inhibitor of IKK2 in neurons,
Dr. Hiroaki Naraba (Iwate Medical University, Japan) for providing the mPGES-1
constructs Caspar Grond-Ginsbach and Annette Biessman (Kopfklinik, Heidelberg,
Germany) for their help in sequencing the constructs, PD Dr. Anne Regnier-
Vigouroux (Deutsche Krebs Forschung Zentrum, Heidelberg, Germany) for
providing microglial cultures and the protocol to prepare them, Prof. Dr. Angelika
Bierhaus, Stoyan Stoyanov and Axel Erhadt for providing RAGEko animals and
sRAGE, Ira Maegla for her help with RT-PCR and cell culture experiments, Dr. Jens
Kleesiek for his help with RT-PCR procedures, Dr. Ioana Inta (Kinderklinik,
I
Acknowledgements

Heidelberg, Germany) for her technical advices concerning cell culture procedures
and for providing the RcCMV-p65 plasmid, Dr. Ming-Fei Lang for the productive and
technical discussions regarding cloning experiments, Dr. Sajjad Muhammad for
providing brain samples and sections from mice subjected to MCAO, Sasidhar
Murikinati for providing brain sections from mice subjected to MCAO, CD11b-DTR
mice and cDNA from microglia, Dr Antje Krenz for translating the summary into
German and all my other colleagues in the research group of Prof. Schwaninger
and in the Institute of Pharmacology, Heidelberg University for their support and
scientifically productive discussions.
Last but not least I would like to express my deepest gratitude to the Egyptian
Government (Ministry of higher education, Cultural and study-mission department,
LX 048) for financing me and for their continuous support during the whole period of
my dissertation.


II
Contents

Contents
I Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 Zusammenfassung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1. Definition and impact of stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.2. Mechanism of damage associated with stroke . . . . . . . . . . . . . . . . 6
1.2.1. Reactive oxygen species in ischemia . . . . . . . . . . . . . . . . . . . . . 7
1.2.2. Adhesion molecules in ischemia . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.2.3. Matrix metalloproteinases in ischemia . . . . . . . . . . . . . . . . . . . . 8
1.2.4. Transcription factors in ischemia . . . . . . . . . . . . . . . . . . . . . . . . 9
1.2.4.a. Nuclear factor kappa B (NF- κB) in ischemia. . . . . . . . . . 9
1.2.4.b. Other transcription factors in ischemia . . . . . . . . . . . . . . 12
1.2.5. Role of cytokines in ischemia . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.2.5.a. Role of TNF in ischemia . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.2.5.b. Role of interleukin-1 (IL-1) family in ischemia . . . . . . . . 14
1.2.5.c. Role of other cytokines in ischemia . . . . . . . . . . . . . . . . . . 15
1.2.6. Role of nitric oxide in ischemia . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.2.7. Role of arachidonic acid and its metabolites in ischemia . . . . . . 15
1.2.7.a. Role of phospholipase A-2 (PLA-2) in ischemia . . . . . . . 17
1.2.7.b. Role of cyclooxygenase (COX) in ischemia . . . . . . . . . . 18
1.2.7.c. Role of prostaglandin E synthase (PGES) in ischemia . 19
1.2.7.d. Role of prostaglandin E (PGE2) in ischemia . . . . . . . . . 19 2
1.2.8. Role of High Mobility Group Box-1 in ischemia . . . . . . . . . . . . . . . 21

1.2.8.a. Role of Receptor for Advanced Glycation End products
in ischemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

25 Aim of the study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
26 2. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.1.1. Preparation of glial cultures (Astrocytes/Microglia) . . . . . . . . . . . . 27
2.1.2. Depletion of microglia from mixed glial cultures . . . . . . . . . . . . . . . 28
2.1.3. Cortical Neuron Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.1.4. Isolation of peritoneal macrophages . . . . . . . . . . . . . . . . . . . . . . . . 30
III
Contents

2.2. Oxygen Glucose Deprivation (OGD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.3. Quantification of cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.3.1. Quantification of cell death and lysis by measurement of lactate
dehydrogenase (LDH) activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.3.2. Detection of cytoplasmic histone-associated-DNA-fragments
32 (mono-and oligonucleosomes) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. Quantification of Prostaglandin E (PGE ) release . . . . . . . . . . . . . . . . 33 2 2
2.5. RNA extraction and reverse transcription . . . . . . . . . . . . . . . . . . . . . . . . 34
2.5.1. RNA extraction from cultured cells . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2.5.2. RNA extraction from brain tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.5.3. Reverse Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.6. Polymerase chain reaction (PCR), Reverse transcription-PCR (RT-
35 PCR) and Real-Time RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.6.1. Real time RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.6.2. RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.6.3. Genotyping of CD11b-DTR mice . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.7. Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.7.1. Anti-Neuronal Nuclei (NeuN)-Staining . . . . . . . . . . . . . . . . . . . . . . . 42
2.7.2. Anti-Glial Fibrillary Acidic Protein (GFAP)-Staining . . . . . . . . . . . . 42
2.7.3. Anti-ionized calcium binding adaptor molecule 1 (Iba-1)-Staining 42
. . .
2.7.4. Anti-High Mobility Group Box-1 protein (HMGB1)-Staining . . . . . 43
2.7.5. Anti-cluster of differentiation molecule 11b (CD11b)-Staining . . . 43

2.7.6. Anti-Receptor for Advanced Glycation Endproducts (RAGE)-
Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.8. Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.8.1. Cloning PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
2.8.2. PCR product purification . . . . . . . . . . . . . . . . . . . . . . . . . . .