Inflammation and Cell Proliferation Following Perinatal Brain Injury

........................................................................................................................ 5 LIST OF ORIGINAL PAPERS......................................................................................... 7 TABLE OF CONTENT ...................................................................................................... 8 ABBREVIATIONS ........................................................................................................... 10 INTRODUCTION ............................................................................................................. 12 Clinical background .................................................................................................... 12 Mechanisms of hypoxic-ischemic perinatal brain injury ................................... 12 Primary injury ............................................................................................................ 13 Secondary injury and secondary energy failure ........................................................ 13 Cell death ....................................................................................................................... 14 Inflammation................................................................................................................. 14 Inflammation in the brain ......................................................................................... 15 Cells involved in inflammation .................................................................................. 15 The complement system ............................................................................................. 16 Activation of the complement system ..................................................................... 17 The classical pathway ................................................................................................ 17 The mannose-binding-lectin pathway ........................................................................ 18 The alternative pathway ............................................................................................. 18 The third complement protein (C3) ......................................................................... 18 Astrocytes and reactive gliosis ................................................................................. 19 Astrocytes .................................................................................................................... 19 Intermediate filaments ............................................................................................... 20 Reactive gliosis in brain injury .................................................................................. 21 Cell proliferation and recovery in the CNS ........................................................... 21 Neurogenesis after brain injury ................................................................................. 21 Cell proliferation after injury in the developing brain .............................................. 22 AIMS OF THIS THESIS ................................................................................................. 23 MATERIALS AND METHODS ...................................................................................... 24 Animals ........................................................................................................................... 24 Hypoxic-ischemic brain injury model ..................................................................... 24 Injections ....................................................................................................................... 25 Bromodeoxyuridine (BrdU) ........................................................................................ 25 Lipopolysaccharide (LPS) .......................................................................................... 25 C3a peptide ................................................................................................................. 26 Tissue preparation ....................................................................................................... 26 Immunohistochemistry............................................................................................... 27 Thionin/ acid fuchsin staining .................................................................................. 27 Immunohistochemical staining of paraffin sections ................................................. 27 Fluorescent staining of paraffin sections ................................................................... 27 Inflammation and Cell Proliferation Following Perinatal Brain Injury 9 Immunohistochemical staining of free floating sections ........................................... 28 Fluorescent staining of free floating sections ............................................................. 28 Histomorphological evaluation ................................................................................ 29 Light microscope cell counting ................................................................................... 29 Confocal scanning laser microscope cell counting ..................................................... 30 Quantitative real-time PCR ....................................................................................... 30 Behavioural experiments ........................................................................................... 32 Fear conditioning with shock-paired tone and light ................................................. 32 Trace fear conditioning with shock-paired tone ........................................................ 32 Fear conditioning with shock-paired tone and additional contextual cues .............. 32 Statistics ......................................................................................................................... 33 RESULTS AND DISCUSSION ...................................................................................... 35 Attenuation of reactive gliosis does not affect the hemisphere or infarct volume, but increases the number of surviving newborn neurons after HI in the developing brain (I) .............................................................................................. 35 Increasing C3a attenuates brain injury in a mouse model of perinatal HI (II) .......................................................................................................................................... 36 Pavlovian fear conditioning is a sensitive method to detect memory impairments following HI injury in neonatal mice (III) ..................................... 37 LPS induced inflammation has different effects on cell survival and proliferation in the immature hippocampus (IV) ................................................. 38 CONCLUDING REMARKS ............................................................................................ 40 ACKNOWLEDGMENTS ................................................................................................. 41 REFERENCES ................................................................................................................. 42