Periventricular white matter damage in the post natal brain in hypoxic conditions

Periventricular white matter damage in the postnatal brain in hypoxic conditions DENG YIYU, MD A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ANATOMY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGMENTS I am deeply indebted to my supervisors, Dr Charanjit Kaur, Associate Professor, Department of Anatomy, National University of Singapore, and Dr Lu Jia, Associate Professor, Defence Medical and Environmental Research Institute, DSO National Laboratories, Singapore, for their constant encouragement, invaluable guidance and infinite patience throughout this study I am very grateful to Professor Ling Eng Ang, former Head of Anatomy Department, National University of Singapore, and Professor Bay Boon Huat, Head of Anatomy Department, National University of Singapore for their constant support and encouragement to me as well as their valuable suggestions to my project, and also for their full support in using the excellent working facilities I would like to acknowledge my gratitude to Dr Viswanathan Sivakumar, Mrs Ng Geok Lan and Mrs Yong Eng Siang for their excellent technical assistance; Mr Yick Tuck Yong for his constant assistance in computer work and Mrs Carolyne Wong, Ms Violet Teo and Mdm Diljit Kour for their secretarial assistance I also wish to thank all staff members and my fellow postgraduate students at Department of Anatomy, National University of Singapore for their assistane one way or another Certainly, without the financial support from the National University of Singapore, in terms of Research Scholarship and National Medical Research Council in terms of a research grant (181-000-65-112 and 181-000-98-112 from NUS) to A/P i Charanjit Kaur, this work would not have been brought to a reality Finally, I am greatly indebted to my wife, Mrs Zhou Yan for her constant encouragement, patience and help during my study ii This thesis is dedicated to my beloved family iii PUBLICATIONS Various portions of the present study have been published or submitted for publication International Journals: Deng Y, Lu J, Sivakumar V, Ling EA, Kaur C Amoeboid microglia in the periventricular white matter induce oligodendrocyte damage through expression of proinflammatory cytokines via MAP kinase signaling pathway in hypoxic neonatal rats Brain Pathol 2008 Jul;18(3):387-400 Deng Y, Lu J, Ling EA, Kaur C Monocyte chemoattractant protein-1 (MCP-1) produced via NF-kappaB signaling pathway mediates of amoeboid microglia in the periventricular white matter in hypoxic neonatal rats Glia 2009 Apr 15; 57(6):604-21 Lu J, Goh SJ, Tng PY, Deng YY, Ling EA, Moochhala S Systemic inflammatory response following acute traumatic brain injury Front Biosci 2009 Jan 1; 14: 3795-813 Deng Y, Lu J, Ling EA, Kaur C Microglia-derived macrophage colony stimulating factor promotes generation of proinflammatory cytokines by astrocytes in the periventricular white matter in the hypoxic neonatal brain Brain Pathol 2010 Mar Deng Y, Lu J, Ling EA, Kaur C Microglia and inflammation in the hypoxic developing brain Revised paper submited to Front Biosci Conference Abstracts: 38th Annual Meeting of the Society for Neuroscience, held on Nov 15 to 19, 2008 in Washington, DC Deng Y, Lu J, Ling EA, Kaur C Monocyte chemoattractant protein-1 (MCP-1) produced via NF-kappa B signaling pathway mediates of amoeboid microglia in the periventricular white matter in hypoxic neonatal rats International Anatomical Sciences and Cell Biology Conference, held on May 26-29, 2010 in Singapore Deng Y, Lu J, Ling EA, Kaur C Microglia-derived macrophage colony stimulating factor promotes generation of proinflammatory cytokines by astrocytes in the periventricular white matter in the hypoxic neonatal brain iv TABLE OF CONTENTS ACKNOWLEDGEMENTS i DEDICATION ….iii PUBLICATIONS iv TABLE OF CONTENTS v ABBREVIATIONS xi SUMMARY xv Chapter 1: Introduction 1.1 Etiology and risk factors associated with PWM……………………………… 1.1.1 Immaturity……………………………… 1.1.2 Hypoxia/ischemia……………………………… 1.1.3 Infection……………………………… 1.1.4 Inflammation……………………………… .5 1.1.5 Vascular factor……………………………… .5 1.1.6 Other risk factors……………………………… 1.2 Pathological changes in the PWMD………………………………………………6 1.3 Oligodendrocytes development, maturity, myelination and injury in the PWMD.7 1.4 Axon injury in the PWMD……………………………………………………….11 1.5 Role of astrocytes in the PWMD…………………………………………………13 1.6 Role of microglia in the PWMD…………………………………………………16 1.6.1 Origin and morphology of microglia………………………………………… 17 v 1.6.2 Properties of microglia ……………………………………………………… 18 1.6.2.1 Phagocytosis………………………………………………………………….18 1.6.2.2 Antigen presentation…………………………………………………………19 1.6.2.3 Proliferation………………………………………………………………….20 1.6.2.4 Migration…………………………………………………………………… 21 1.6.2.5 Generation of reactive oxygen species (ROS) and nitrogen intermediates… 22 1.6.2.6 Release of cytokines and chemokines……………………………………… 24 1.6.2.6.1 TNF-a and its receptors…………………………………………………….24 1.6.2.6.2 IL-1 and its receptors……………………………………………………….29 1.6.2.6.3 Macrophage-colony stimulating factor ….……………………………… 30 1.6.2.6.4 Monocyte chemoattractant protein-1… ………………………………… 30 1.7 Aim of this study…………………………………………………………………32 1.7.1 To examine the role of AMC in the PWMD………………………………… 33 1.7.2 To examine if MCP-1 mediates migration of AMC in the PWM in hypoxic neonatal rats…………………………………………………………………… ……34 1.7.3 To study the role of M-CSF produced by AMC in generation of TNF-α and IL-1β by astrocytes in the PWM in hypoxic neonatal rats….……………………… 35 Chapter 2: Materials and Methods 37 2.1 Animals………………………………………………………………………… 38 2.2 Mixed Glial Cell Culture ……………………………………………………… 39 2.2.1 Materials……………………………………………………………………….39 2.2.2 Procedure…………………………………………………………………… 40 vi 2.1.2.1 Removal of Brain Tissue…………………………………………………….40 2.2.2.2 Mechanical dissociation of brain tissue…………………………………… 40 2.2.2.3 Enzymatic digestion…………………………………………………………41 2.2.3 Microglia purification…………………………………………………………41 2.2.4 Astrocytes purification……………………………………………………… 42 2.3 Treatment of Microglial Cell Culture…………………………………………….43 2.4 Treatment of Astrocytes Culture…………………………………………………44 2.5 RNA Isolation and Real time reverse transcription-polymerase chain reaction (RT-PCR)…………………………………………………………………………… 45 2.5.1 Materials……………………………………………………………………….45 2.5.2 Procedure……………………………………………………………………….46 2.5.2.1 Extraction of total RNA…………………………………………………… 46 2.5.2.2 cDNA Synthesis…………………………………………………………… 46 2.5.2.3 Real time RT-PCR……………………………………………………………47 2.5.2.4 Detection of PCR product……………………………………………………49 2.6 Western Blot assay………………………………………………………………50 2.6.1 Materials……………………………………………………………………….50 2.6.2 Procedure………………………………………………………………………53 2.7 Immunofluorescence labeling………………………………………………… 55 2.7.1 Materials……………………………………………………………………….55 2.7.2 Procedure for double immunoflourescence …………………………………56 2.7.2.1 Double immunoflourescence in vivo…………………………………………56 vii 2.7.2.2 Double immunoflourescence in vitro………………… …………………….57 2.8 Electron microscopy…………………………………………………………… 59 2.9 Detection of oligodendrocyte apoptosis by fluorescence terminal deoxynucleotidyl transferase (Tdt)-mediated dUTP nick end labelling (TUNEL) assay ……………………………………………………………………………………… 59 2.10 Intracerebral stereotactic injection of MCP-1………………………………….60 2.11 Cell counting and proliferation of AMC by lectin and 5-bromo-2’-deoxyuridine (BrdU) labeling………………………………………………………………………61 2.12 Cell counting of AMC following MCP-1 injection labeled with lectin or OX-42……………………………………………………………………………… 62 2.13 ELISA………………………………………………………………………… 63 2.13.1 Materials…………………………………………………………………… 63 2.13.2 Analysis of MCP-1 by ELISA……………………………………………… 64 2.14 Chemotaxis…………………………………………………………………… 64 2.14.1 Materials…………………………………………………………………… 64 2.14.2 Procedure…………………………………………………………………… 65 2.15 Statistical Analysis…………………………………………………………… 66 Chapter 3: Results……………………………………………………………….67 3.1 Real time RT-PCR analysis of TNF-α, IL-1β, TNF-R1 and IL-1R1, M-CSF, CSF-1R, MCP-1 and CCR2 mRNA expression in the PWM…… ………………….68 3.2 Western blotting or ELISA analysis of TNF-α, IL-1β, TNF-R1 and IL-1R1, M-CSF, CSF-1R, MCP-1 and CCR2 protein expression in the PWM……………………… 69 viii 3.3 Cellular localization of TNF-α, IL-1β, TNF-R1 and IL-1R1, M-CSF, CSF-1R, MCP-1 and CCR2 protein expression in the PWM by double labeling…………… 70 3.4 MBP and NF-200 protein expression in the PWM …………………………… 72 3.5 Apoptosis of oligodendrocytes in the PWM …………………………………….73 3.6 Ultrastructural observations…………………………………………………… 73 3.7 Increase in cell numbers of AMC in the PWM in hypoxic neonatal rats ……… 74 3.8 MCP-1 induced microglial migration in vivo…………………………… …….75 3.9 mRNA and protein expression of TNF-α, IL-1β, M-CSF and MCP-1 in activated microglia under hypoxic conditions………………………………………………….75 3.10 Hypoxia induced the TNF-α and IL-β production via activation of MAP kinase pathway in activated microglia ………………………………………………………77 3.11 Hypoxia induced MCP-1 production via activation of NF-kappaB signaling pathway in microglia ……………………………………………………………… 78 3.12 TNF-α, IL-1β and CSF-1R mRNA and protein expression in activated astrocytes after M-CSF treatment ………………………………………………………………79 3.13 Increased TNF-α and IL-β production in activated astrocytes after M-CSF treatment was via activation of MAP kinase pathway ………………………………80 3.14 Migration of microglia to medium derived from microglial culture subjected to hypoxia………………………….……………………………………………………81 Chapter 4: Discussion 83 4.1 Microglia are activated and induce a robust and persistent inflammatory response in the PWM in hypoxic neonatal rats ……………………………………………….84 ix A Control Lectin B Control BrdU C Control Merge D Hypoxia 3h Lectin E Hypoxia 3h BrdU F Hypoxia 3h Merge G Control Lectin H Control BrdU I Control Merge K Hypoxia 24h BrdU 16 * N 14 12 10 * control hypoxia 3h 24h P ercen tag e o f m icro g lia p ro liferatio n in th e PW M M Number of microglia in the PWM/mm2 J Hypoxia 24h Lectin Fig.20 L Hypoxia 24h Merge 0.18 0.16 0.14 0.12 0.1 control hypoxia 0.08 0.06 0.04 0.02 3h 24h 170 B Control BrdU C Control Merge D Hypoxia 3d Lectin E Hypoxia 3d BrdU F Hypoxia 3d Merge G Control Lectin H Control BrdU I Control Merge J Hypoxia 7d Lectin K Hypoxia 7d BrdU L Hypoxia 7d Merge N M Number of microglia in the PWM/mm2 16 * 14 * 12 10 control hypoxia 3d 7d Percentage of microglia proliferation in the PW M A Control Lectin Fig.21 0.18 * 0.16 0.14 0.12 0.1 control hypoxia 0.08 0.06 0.04 0.02 3d 7d 172 A Control B Ipsilateral C Contralateral E Ipsilateral F Contralateral G MCP-1 injection 8h H Ipsilateral I Contralateral J M icroglial cells in the PW M /per view D 0.9% NaCl injection 8h 80 * 70 60 50 Contralateral Ipsilateral 40 30 20 10 Sham-operated 0.9% NaCl injection Fig.22 MCP-1 injection 174 B Ipsilateral OX42 C Contralateral OX42 D 0.9% NaCl injection 8h E Ipsilateral OX42 F Contralateral OX42 G MCP-1 injection 8h H Ipsilateral OX42 I Contralateral OX42 J M icroglial cells in the PW /per view M A Control 100 * 90 80 70 60 Contralateral Ipsilateral 50 40 30 20 10 Sham-operated 0.9% NaCl injection Fig.23 MCP-1 injection 176 A B TNF-αmRNA Fold change Fold change * * 12 * IL-1βmRNA 14 * * * 10 * * 2 0 control hypo 1h hypo 2h hypo 4h control hypo 6h C D hypo 1h hypo 2h 12 * Fold change M-CSF mRNA * * * 10 * fold change hypo 6h MCP-1 mRNA 14 M-CSF mRNA hypo 4h * * 2 control 1h 2h Hypoxic time 4h 6h control hypo 1h hypo 2h hypo 4h hypo 6h Fig.24 178 A Control Hypoxia 2h 4h 1h 6h TNF-α 30kDa IL-1β 17kDa M-CSF 18.5kDa β-actin 42kDa B C TNF-α * * * * * * Optical density Optical density IL-1β control 1h 2h 4h 6h control Hypoxic time M-CSF E Optical density 400 * 3.5 * 450 Amount of MCP-1 released (pg/ml) D 4.5 350 1h 2h Hypoxic time ELISA for MCP-1 200 1.5 * * 150 * * * 250 * 6h C2 hypo 4h 300 2.5 4h * * 100 0.5 50 control 1h 2h Hypoxic time 4h 6h * control hypo 1h hypo 2h hypo 4h hypo 6h Fig.25 180 A Control Lectin B Control TNF-ɑ C Control Merge E Hypoxia 4h TNF-ɑ F Hypoxia 4h Merge G Control Lectin H Control IL-1β I Control Merge J Hypoxia 4h Lectin K Hypoxia 4h IL-1β L Hypoxia 4h Merge D Hypoxia 4h Lectin Fig.26 182 A Control Lectin B Control M-CSF C Control Merge D Hypoxia 4h Lectin E Hypoxia 4h M-CSF F Hypoxia 4h Merge G Control Lectin H Control MCP-1 I Control Merge K Hypoxia 4h MCP-1 L Hypoxia 4h Merge A2 hypo 4h J Hypoxia 4h Lectin Fig.27 184 Hypoxia 1h 2h ½h control control hypoxia 4h hypoxia 4h+JNK inhibitor 4h A Phos-JNK Total-JNK D E TNF-α IL-1β β-actin B Phos-p38 control hypoxia 4h hypoxia 4h+p38 inhibitor Total-p38 TNF-α Phos-ERK C F G IL-1β β-actin Total-ERK H * I Phos-JNK 12 * J Phos-p38 Optical density Optical density Optical density Phos-ERK 16 14 12 ** 10 ** ** * 18 10 20 0 4.5 3.5 2.5 1.5 0.5 1h hypoxic time SP600125 TNF-α Optical density 4.5 3.5 2.5 1.5 0.5 4h * control * 1/2h 1h hypoxic time L 2h 3.5 4h control 1/2h 1h hypoxic time 2h 4h * SB203580 TNF-α 2.5 1.5 0.5 control M 2h Optical density Optical density K 1/2h hypoxia 4h * SP600125 IL-1β control hypoxia+SP N Optical density control hypoxia 4h SB203580 IL-1β hypoxia+SB * control hypoxia 4h control hypoxia+SP hypoxia 4h hypoxia+SB Fig.28 186 B Control phos-c-JUN C Control Merge D Hypoxia DAPI E Hypoxia Phos-c-JUN F Hypoxia Merge G Percentage of c-JUN positive microglial cells A Control DAPI * 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 control hypo 45min Fig.29 188 Hypoxia Control 15min 30min 1h 2h A Phos -NF-kappaB B NF-kappa B C Phos - Ikappa B D Ikappa B β-actin Phos-NF- kappaB E NF- kappaB F 0.25 * * 0.6 * 0.1 0.05 15min 30min hypoxic time 1h * 0.4 * 0.3 0.2 0.1 2h control G 0.7 H Phos-IkappaB 1.5 0.3 * optical density 0.5 ** ** ** ** 0.75 * 0.2 0.5 0.25 0.1 hypoxic time 1h 2h 30min hypoxic time 1h 2h ELISA 500 450 400 350 300 250 200 150 * 100 50 2h 30min 1h 15min 30 mi n control 15 mi n co nt ro l optical density 1.25 * 15min I IkappaB 0.6 0.4 * Amount of MCP-1 released (ng/ml) control * 0.5 * 0.15 optical density optical density 0.2 control hypo 4h hypo 4h+ BAY hypoxic time Fig.30 190 A B TNF-αmRNA * * 6 * Fold change Fol c nge d IL-1βmRNA * * * 2 1 0 control C 3h 6h 12h M-CSF treatment time 24h control 3h 6h 12h M-CSF treatment time 24h CSF-1R mRNA * Fold change * * * control 3h 6h 12h M-CSF treatment time 24h M-CSF treatment D Control 3h 6h 12h 24h TNF-α IL-1β CSF-1R β-actin E 10 Optical density * * * IL-1β * 12 Optical density * 14 F TNF-α 10 * * * 2 control G 3h 6h M-CSF treatment time 12h 24h control 3h 6h 12h M-CSF treatment time 24h CSF-1R 3.5 * Optical density 2.5 1.5 * * * 0.5 control 3h 6h 12h M-CSF treatment time 24h Fig.31 192 A Control GFAP B Control TNF-α C Control Merge D M-CSF 12h GFAP E M-CSF 12h TNF-α F M-CSF 12h Merge G Control GFAP H Control IL-1β I control Merge J M-CSF 12h GFAP K M-CSF 12h IL-1β L M-CSF 12h Merge M Control GFAP N Control CSF-1R O Control Merge P M-CSF 12h GFAP Q M-CSF 12h CSF-1R R M-CSF 12h Merge Fig.32 194 Control M-CSF treatment 15min 30min 1h control M-CSF 3h M-CSF 3h+JNK inhibitor 2h A Phos-JNK Total-JNK B D E TNF-α IL-1β β-actin Phos-p38 control M-CSF 3h M-CSF 3h+p38 inhibitor Total-p38 C F G Phos-ERK Total-ERK H 25 I Phos-JNK ns ns 1h 2h Optical density Optical density Phos-p38 4.5 * 20 TNF-α IL-1β β-actin 15 * 10 ns ns 15min 3.5 30min M-CSF treatment time 2.5 1.5 0.5 control J 15min 30min M-CSF treatment time 2h control Phos-ERK 14 * 12 K SP600125 14 TNF-α 12 * 10 * 10 Optical density Optical density 1h 8 2 control 15min 30min M-CSF treatment time control IL-1β * 14 M-CSF 3h N SB203580 TNF-α 6 Optical density 12 10 M-CSF 3h+SP SB203580 ns 16 Optical density ns IL-1β 5 Optical density 18 2h M SP600125 L 1h 4 2 0 control M-CSF 3h M-CSF 3h+SP control control Fig.33 M-CSF 3h M-CSF 3h M-CSF 3h+SB M-CSF 3h+SB 196 A DMEM Control Medium Hypo 4h+antiMCP-1 Hypo 4h+antiserum * 450 B Hypo 4h * * Migration of Microglia (%) 400 350 300 250 200 150 100 50 DMEM control hypoxia 4h Hypoxia 4h + Hypoxia 4h + antiMCP-1 antiserum Fig.34 198 ... astrocytes in the periventricular white matter in the hypoxic neonatal brain Brain Pathol 2010 Mar Deng Y, Lu J, Ling EA, Kaur C Microglia and inflammation in the hypoxic developing brain Revised... myelin sheath Axons in the neonatal brain are not myelinated Myelination of axons in the periventricular white matter (PWM) of developing rat brain was first observed at the end of the first postnatal... VEGF may induce inflammatory response in the hypoxic neonatal brain leading to PWMD (Min et al 2005) 1.6 Role of microglia in the PWMD AMC, present in large numbers in the developing PWM (Ling and