Pro- und anti-inflammatorische Aspekte in der Pathogenese der Offenwinkelglaukome [Elektronische Ressource] = Pro- and anti-inflammatory aspects of the pathogenesis of open angle glaucoma / vorgelegt von Christian Manfred Hammer

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Pro und Antiinflammatorische Aspekte in der Pathogenese der Offenwinkelglaukome Pro and Antiinflammatory Aspects of the Pathogenesis of Open Angle Glaucoma Der Naturwissenschaftlichen Fakultät der FriedrichAlexanderUniversität ErlangenNürnberg zur Erlangung des Doktorgrades Dr. rer. nat. vorgelegt von Christian Manfred Hammer aus Würzburg Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der FriedrichAlexanderUniversität ErlangenNürnberg Tag der mündlichen Prüfung: 23.06.2010 Vorsitzender der Promotionskommission: Prof. Dr. Eberhard Bänsch Erstberichterstatter: Prof. Dr. J. H. Brandstätter Zweitberichterstatter: Prof. Dr. E. LütjenDrecoll This dissertation and the future that is based on it are dedicated to Wilbert, Erbsbert and Froffl and to my wife Sonja, who showed me the right way. “for the times, they are achangin’!” Bob Dylan Table of Contents 1 TABLE OF CONTENTS ABBREVIATIONS ................................................................................................................... 3 ZUSAMMENFASSUNG .........................................................................................................5 ABSTRACT..............................................................
Publié le : vendredi 1 janvier 2010
Lecture(s) : 18
Tags :
Source : D-NB.INFO/1004842325/34
Nombre de pages : 122
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Pro und Antiinflammatorische Aspekte in der
Pathogenese der Offenwinkelglaukome



Pro and Antiinflammatory Aspects of the
Pathogenesis of Open Angle Glaucoma



Der Naturwissenschaftlichen Fakultät
der FriedrichAlexanderUniversität
ErlangenNürnberg

zur

Erlangung des Doktorgrades Dr. rer. nat.



vorgelegt von



Christian Manfred Hammer
aus Würzburg

Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät
der FriedrichAlexanderUniversität ErlangenNürnberg















Tag der mündlichen Prüfung: 23.06.2010








Vorsitzender der Promotionskommission: Prof. Dr. Eberhard Bänsch
Erstberichterstatter: Prof. Dr. J. H. Brandstätter
Zweitberichterstatter: Prof. Dr. E. LütjenDrecoll



This dissertation
and the future that is based on it
are dedicated to





Wilbert, Erbsbert
and Froffl





and to my wife Sonja,
who showed me the right way.







“for the times, they are achangin’!”
Bob Dylan

Table of Contents 1

TABLE OF CONTENTS

ABBREVIATIONS ................................................................................................................... 3
ZUSAMMENFASSUNG .........................................................................................................5
ABSTRACT...............................................................................................................................9


1. INTRODUCTION ........................................................................................................... 13
1.1. Glaucoma............................................................................................................... 13
1.1.1. Open angle glaucoma ................................................................................... 13
1.1.2. Steroidinduced secondary glaucoma ........................................................ 20
1.1.3. Uveitisassociated secondary glaucoma ................................................... 22
1.1.4. Endothelial leukocyte adhesion molecule1 .............................................. 23
1.2. Trabecular meshwork......................................................................................... 25
1.2.1. General morphology of the TM .................................................................... 25
1.2.2. Functional aspects of the TM....................................................................... 28
1.2.3. Glaucomatous alterations of the TM........................................................... 29
1.3. Concept of this study ......................................................................................... 32
1.3.1. Morphological changes in steroidtreated bovine eyes ........................... 32
1.3.2. Evaluation of uveitic glaucoma trabeculectomy specimens ................... 32
1.3.3. Putative IL1/ELAM1 stress response in the TM ..................................... 32

2. METHODS ...................................................................................................................... 34
2.1. Steroidtreated bovine eyes ............................................................................. 34
2.1.1. Animal eyes and steroid treatment ............................................................. 34
2.1.2. Fixation and preparation of the eyes .......................................................... 35
2.1.3. Histology and electronmicroscopy .............................................................. 35
2.1.4. Immunohistofluorescence ............................................................................ 36
2.1.5. Immunoelectronmicroscopy ........................................................................ 37
2.1.6. Semiquantitative and quantitative analyses .............................................. 37
2.2. Uveitisassociated secondary glaucoma specimens ................................ 39
2.2.1. Patients ........................................................................................................... 39
2.2.2. Histological and ultrastructural analysis ..................................................... 40
2.2.3. Quantitative evaluation of the cribriform TM ............................................. 41

Table of Contents 2


2.3. IL1/ELAM1 project ............................................................................................ 41
2.3.1. Survey of experiments .................................................................................. 41
2.3.2. Anterior eye perfusion culture ...................................................................... 47
2.3.3. RNA analysis .................................................................................................. 51
2.3.4. Protein analysis .............................................................................................. 55

3. RESULTS ....................................................................................................................... 57
3.1. Morphological findings in steroidtreated bovine eyes ............................ 57
3.1.1. Calf and Braford cow eyes ........................................................................... 57
3.1.2. Steroidtreated eyes ...................................................................................... 59
3.1.3. Contralateral control eyes ............................................................................ 61
3.1.4. Staining for typeVIcollagen ........................................................................ 62
3.1.5. Semiquantitative and quantitative analyses .............................................. 63
3.2. Morphological observations in uveitisglaucoma specimens ................ 65
3.2.1. Fuchs’ Heterochromic Cyclitis (FHC) ......................................................... 65
3.2.2. Juvenile Idiopathic Arthritis (JIA) ................................................................. 68
3.2.3. Herpes zoster uveitis..................................................................................... 69
3.3. Cytokine/ELAM1 experiments ........................................................................ 71
3.3.1. ELAM1 staining............................................................................................. 71
3.3.2. IL1α perfusion culture .................................................................................. 72
3.3.3. IL1β perfusion culture .................................................................................. 76
3.3.4. IL6 perfusion culture .................................................................................... 78
3.3.5. IL1α/TGFβ2 perfusion culture ................................................................... 80
3.3.6. Cell culture experiments ............................................................................... 82

4. DISCUSSION ................................................................................................................. 85
4.1. Steroid treatment of bovine eyes ................................................................ 85
4.2. Uveitic glaucoma specimens ....................................................................... 88
4.3. ELAM1 induction and IOP regulation ....................................................... 90
4.4. Synopsis ............................................................................................................ 94

REFERENCES....................................................................................................................... 97
ACKNOWLEDGMENTS .................................................................................................... 112
REGISTER ........................................................................................................................... 113
CHEMICALS & REAGENTS..............................................................................................114 Abbreviations 3

ABBREVIATIONS
AH Aqueous humour
bp Base pairs
BSA Bovine serum albumine
CAPS 3cyclohexylamino1propanesulfonic acid
cDNA Complementary Desoxyribonucleic acid
DEPC Diethylpyrocarbonate
DMEM Dulbecco’s Modified Eagle’s Medium
DOC Deoxycholic acid
DTT Dithiothreitol
ECM Extracellular material
EDTA Ethylenediaminetetraacetate
ELAM1 Endothelial leukocyte adhesion molecule1
FHC Fuchs’ Heterochromic Cyclitis
GAPDH Glycerinaldehyde 3phosphate dehydrogenase
h Hours
IL1α Interleukin1α
IL1β Interleukin1β
IL6 Interleukin6
IOP Intraocular pressure
JIA Juvenile Idiopathic Arthritis
kD Kilodalton
min Minutes
mRNA Messengerribonucleic acid
ON Optic nerve
PAI1 Plasminogen activator inhibitor1
PBS Phosphatebuffered saline
PBST Phosphatebuffered saline + 0.2% TritonX100
PMSF Phenylmethylsulfonylfluoride
POAG Primary open angle glaucoma
qPCR Quantitative real time PCR
rcf Real centrifugation force
rER Rough endoplasmatic reticulum
rpm Rounds per minute
RT Room temperature
RTPCR Reverse transcriptase polymerase chain reaction
SC Schlemm’s Canal
SD Standard deviation
SDS Sodium dodecyl sulfate
sec Seconds
SELP Selectin P
SG Steroid glaucoma Abbreviations 4

TBE Tris base/boric acid/EDTA
TBS Trisbuffered saline
TBST Trisbuffered saline + 0.2% TritonX100
TGFβ2 Transforming growth factor β2
TM Trabecular meshwork
VL Vessel loop

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