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Informations
Publié par | justus-liebig-universitat_giessen |
Publié le | 01 janvier 2009 |
Nombre de lectures | 15 |
Langue | English |
Extrait
Characterization of the ROS Production in
Ischemia/Reperfusion-induced Lung Injury
Inaugural Dissertation
submitted to the
Faculty of Medicine
in partial fulfilment of the requirements
for the PhD-Degree
of the Faculties of Veterinary Medicine and Medicine
of the Justus Liebig University Giessen
by
Akylbek Sydykov
of
Osh, Kyrgyz Republic
Giessen 2009
From the Medical Clinic II, University of Giessen Lung Centre
Chairman: Werner Seeger, Prof., M.D.
of the Faculty of Medicine of the Justus Liebig University Giessen
First Supervisor and Committee Member: Prof. Dr. Ardeschir Ghofrani
Second Supervisor and Committee Member: Prof. David B. Pearse, MD
Committee Member (Chair): Prof. Dr. Wolfgang Kummer
Committee Member: Prof. Dr. Joachim Roth
thDate of Doctoral Defence: 27 of October, 2009
Index of contents
Index of contents
Index of contents……………………………………………………………………………...i
Index of tables……………………………………………………………………………….iv
Index of figures…………………………………………………………………………….....v
1. Introduction…………………………………………………………………...……….1
1.1.Pathophysiology of ischemia/reperfusion lung injury.……………....….………………..1
1.1.1. Definitions……………………………………….……………….…….……………..1
1.1.2. Organ ischemia…………………..………….…….….….…………………………...2
1.1.3. Reperfusion of the ischemic organ…………………….…..…………………………2
1.1.4. Leukocytes and ischemia/reperfusion………………………………………………..3
1.1.5. Endothelial cells and ischemia/reperfusion…………………………..………………4
1.2.Evaluation of lung injury in isolated lungs.……………………..……………….……….5
1.2.1. Measurement of pulmonary edema…………………………………………………...5
1.2.2. Permeability measurements………………….……………………………………….5
1.3.Oxidative stress in lung ischemia/reperfusion……………….…………………………...7
1.3.1. Oxidative stress and biology of reactive oxygen species………………..……………8
1.3.2. NADPH oxidases in ischemia/reperfusion………………………………..………….8
1.3.2.1. NADPH oxidase family……….…………………………………..…………...10
1.3.2.2. Phagocytic NADPH oxidase.…….…………………………………………….10
1.3.2.3. Endothelial NADPH oxidase…………………………………………………..11
1.3.3. Inhibitors of NADPH oxidase……………………………………………………….11
1.3.4. Electron spin resonance (ESR) spectroscopy……………………………………….13
1.4.Aim of the study………………………………………………………………………...15
2. Experimental protocols…………………………………..………………………...16
2.1.Ischemia/reperfusion protocols...……………………………………………………......16
2.1.1. Isolated lung preparations……………….………………………………..…………16
2.1.2. In vivo ischemia-reperfusion………………………………………………………..16
2.2.Measurement of vascular permeability and lung weight gain.…………………….........17
2.3.Measurements of ROS production………..…………..………..………………………..18
3. Materials………………………………….………………….……………….….……19
3.1.Chemicals, injecting solutions and drugs.………………………….……………...........19
3.2.Consumables………………………………………..………..………………………….20
i Index of contents
3.3.Composition of Krebs-Henseleit solution………………………………………………22
3.4.Systems and machines for animal experiments.………….……………………………..22
3.5.Software…………………………………………………………………………………23
4. Methods…………..…………………………………………………….……………...24
4.1.Animals………………..…………………………………………………………...........24
4.2.Isolated ventilated and perfused lungs……………..……………………………………24
4.2.1. Isolation, perfusion and ventilation of rabbit lungs.………..………………….……25
4.2.2. Isolation, perfusion and ventilation of mouse lungs.………..………………………26
4.2.3. Vascular compliance measurements………………..…………………..…………...27
4.2.4. Vascular permeability measurements………………..……………………………...28
4.2.5. Assessment of pulmonary edema……………………………………………………28
4.3.Western Blot Assay ………………………………………………….…………............29
4.4.Measurement of nitrite and nitrate in perfusate…………………………………………29
4.5.Ischemia-reperfusion in living mice…………………………………………………….30
4.6.Wet-to-dry lung weight ratio……………………………………………………………30
4.7.Generation of chimeric mice……………………………………………………………31
4.7.1. Technique of bone marrow transplantation…………….……….…………………..31
4.7.2. Types of chimeric mice………………………….…………….…………………….32
4.8.Genotyping of chimeric mice…………………………………………………………...33
4.8.1. Isolation of genomic DNA from peripheral blood leukocytes……….……………...33
4.8.2. ic DNA from endothelial cells……………………………….….34
4.8.3. Polymerase chain reaction…………………………………………………………..34
4.8.4. DNA agarose gel electrophoresis……………………………………………………35
4.9.Measurement of intravascular ROS release…..……………………….………………...36
4.9.1. Perfusion buffer preparation……………………………...…………………………36
4.9.2. Spin probe preparation………………………………………………………………37
4.9.3. ESR spectroscopy settings……………………………...…………………………...37
4.9.4. ROS measurements………………………………………………………………….37
4.10.Endothelial cell culture………………….…..……………………….………………...38
4.10.1. Isolation of human umbilical vein endothelial cells from umbilical cord…………..38
4.10.2. Anoxia-reoxygenation protocol in endothelial cell culture…………………………39
4.10.3. ROS measurement in endothelial cell anoxia-reoxygenation……………………….39
4.11.Data analysis………………………………………………………………..……….....40
5. Results…………………………………………………………………………….….....41
ii Index of contents
5.1. Effects of ischemia and reperfusion on rabbit lungs………………………..…….....32
5.1.1. Hemodynamic data………………………………………………………………….41
5.1.2. Vascular compliance………………………………………………………………...42
5.1.3. Vascular permeability……………………………………………………………….43
5.1.4. Pulmonary edema formation………….……………………………………………..44
5.1.5. Intravascular ROS release……………………………………………………...........45
5.2. Effects of ischemia and reperfusion on lungs from WT and Nox2 KO mice…….....46
5.2.1. Hemodynamic data………………………………………………………………….46
5.2.2. Vascular compliance………………………………………………………………...47
5.2.3. Vascular permeability……………………………………………………………….48
5.2.4. Pulmonary edema formation………….……………………………………………..49
5.2.5. Intravascular ROS release……………………………………………………...........50
5.2.6. Expression of different NOS isoforms in lungs from WT and Nox2 KO mice……..51
5.2.7. NO production in lungs from WT and Nox2 KO mice……………………………..52
5.3. Effects of lung ischemia and reperfusion in living WT and Nox2 KO mice………..53
5.4. Characterization of chimeric mice…………………………………………………..54
5.5. Effects of ischemia and reperfusion on lungs from chimeric mice……………..…..55
5.5.1. Hemodynamic data………………………………………………………………….55
5.5.2. Vascular compliance………………………………………………………………..56
5.5.3. Vascular permeability.………………………………………………………………57
5.5.4. Pulmonary edema formation ………………………………………………………..58
5.5.5. Intravascular ROS release…………………………………………………………...59
5.6. Effects of anoxia-reoxygenation on endothelial ROS production…………………..60
6. Discussion……………………………………………………………………..…..…..61
6.1.Role of NADPH oxidase in ischemia/reperfusion induced lung injury………………...61
6.2.Endothelial not granulocytic NADPH oxidase plays a role in I/R lung injury…………65
7. Summary…………….……………….………………………………………………..69
8. Zusammenfassung..………………………………………………...…………….....70
9. References………..………………………………………….………………………...72
List of abbreviations………..…………………………………………….…………....88
Appendix………………………………………………………………………………….90
A. Acknowledgments………………………………………………….………...……...90
B. Curriculum vitae………………………………………..……………..………..……91
C. Statement/Erklärung an Eides Statt..………………………………………………...96
iii Index of contents
Index of tables
1. Tissue distribution of Nox enzymes …..……………………..……......……………...…..9
2. Nucleotide sequences of primers used for PCR……………………………………..…..34
3. Composition of 25 µl PCR mix for one sample………………………………………….35
4. Cycling conditions for PCR……………………………………………………………...35
5. Parameters of ESR spectroscopy……………...…………………………………………37
6. Pulmonary artery pressure in rabbit lungs……………………………………………….41
7. Vascular compliance in rabbit lungs……………………………………………………..42
8. Pulmonary artery pressure in WT and Nox2 KO mouse lungs………………………….46
9. Vascular compliance in WT and Nox2 KO mouse lungs………………………………..47
10. Pulmonary artery pressure in chimeric mouse lungs………………………………..55
11. Vascular compliance in chimeric mouse lungs…………….………………………..56
Index of figures
1. Activation of ROS generation by assembly of Phox regulatory proteins in phagocytes....10
2. Reaction of CPH with superoxide………………………………………………………...14
3. Representative original tracings of the isolated perfused mouse lung experiment……….17
4. Isolated perfused and ventilated mouse lung….……………...…………………….18
5. Schematic representation of the experimental set-up of the isolated perfused mouse
lung…………………………………………………………….…………………………27
6. Schematic representation of chimeric mice generation………...…...……..……………..31
7. Schematic representation of Nox2