universidade de lisboa faculdade de...

UNIVERSIDADE DE LISBOA

FACULDADE DE FARMÁCIA

MOLECULAR MECHANISMS OF MICROGLIA REACTIVITY TO BILIRUBIN: EVALUATION OF POTENTIAL

NEUROLOGICAL EFFECTS

Sandra Isabel Leitão da Silva

DOUTORAMENTO EM FARMÁCIA (Biologia Celular e Molecular)

2010

UNIVERSIDADE DE LISBOA

FACULDADE DE FARMÁCIA



Sandra Isabel Leitão da Silva

Research advisors: Dora Maria Tuna de Oliveira Brites, PhD. Rui Fernando Marques da Silva, PhD.

DOUTORAMENTO EM FARMÁCIA (Biologia Celular e Molecular)

2010



MECANISMOS MOLECULARES DE REACTIVIDADE DA

MICROGLIA À BILIRRUBINA: AVALIAÇÃO DE POTENCIAIS

EFEITOS NEUROLÓGICOS

Dissertação apresentada à Faculdade de Farmácia da Universidade de Lisboa para obtenção do grau de Doutor em Farmácia (Biologia Celular e Molecular)

Sandra Isabel Leitão da Silva 2010

Para a elaboração da presente tese de doutoramento foram usados integralmente como

capítulos, artigos científicos publicados, ou submetidos para publicação, em revistas

científicas internacionais indexadas. Estes trabalhos foram realizados em colaboração

com os seguintes autores: Ana Rita Vaz, Catarina Osório, Andreia Barateiro, Adelaide

Fernandes, Ana Sofia Falcão, Maria José Diógenes, Maria Alexandra Brito, Nico van

Rooijen, Ana Sebastião, Rui Silva e Dora Brites.

De acordo com o disposto no ponto 1 do artigo nº41 do Regulamento de Estudos Pós-

Graduados da Universidade de Lisboa, deliberação nº 93/2006, publicada em Diário da

República – II Série nº 153 – 5 de Julho de 2003, a Autora desta dissertação declara

que participou na concepção e execução do trabalho experimental, na interpretação dos

resultados obtidos e na redacção dos manuscritos.

Os estudos apresentados nesta dissertação foram realizados no grupo de investigação

“Neuron Glia Biology in Health & Disease”, Research Institute for Medicines and

Pharmaceutical Sciences (iMed.UL), Faculdade de Farmácia da Universidade de Lisboa.

Parte do trabalho foi também realizado no Departamento de Investigação Cellular

Neuroscience do Max-Delbrück Centrum em Berlim, Alemanha, sob a orientação do

Professor Doutor Helmut Kettenmann.

O trabalho foi subsidiado pelos projectos FCT-POCTI/SAU/MMO/55955/2004 e FCT-

PTDC/SAU-NEU/64385/2006 concedidos à Professora Doutora Dora Brites pela

Fundação para a Ciência e Tecnologia (FCT), sendo que a Autora usufruiu de uma

bolsa de Doutoramento (SFRH/BD/30326/2006) concedida pela FCT, Lisboa, Portugal.

Para o meu Pai

Acknowledgements/Agradecimentos As minhas primeiras palavras de agradecimento vão naturalmente para a

Professora Doutora Dora Brites, orientadora deste doutoramento. Gostaria de lhe

agradecer o facto de me ter aberto as portas do mundo da ciência e por tão bem me ter

guiado nos seus trilhos. Gostaria de salientar as suas notáveis qualidades científicas e o

seu enorme espírito crítico, bem como o seu sentido de justiça e igualdade. Os seus

elevados padrões de rigor científico e o seu constante encorajamento ao longo deste

caminho fizeram-me acreditar que podia chegar ao fim e, mais importante do que isso,

que era sempre possível fazer mais e melhor. Como já lhe tenho dito muitas vezes, o

seu olhar consegue sempre ver mais além! Os seus ensinamentos ser-me-ão úteis ao

longo da vida académica e pessoal e não esquecerei o tempo que passei a trabalhar ao

seu lado nem o facto de ter depositado em mim a confiança para desempenhar esta

tarefa. Só espero ter estado à altura das suas expectativas!

Ao Professor Doutor Rui Silva, co-orientador desta tese, quero agradecer a

constante ajuda e encorajamento para superar as dificuldades que foram surgindo ao

longo deste percurso académico. O seu olhar crítico sobre as questões essenciais foram

extremamente valiosas para a concretização deste trabalho. Quero também agradecer a

preocupação e carinho que sempre demonstrou para comigo e a forma como sempre

me conseguia fazer sorrir mesmo quando tudo corria mal!

À Professora Doutora Alexandra Brito gostaria de agradecer por, apesar de não

ter uma responsabilidade directa sobre o meu trabalho, ter sempre demonstrado

interesse e disponibilidade para me ajudar. Os seus conselhos científicos foram

pertinentes, assertivos e de extrema validade. Gostaria também de lhe agradecer a

amizade que desenvolvemos ao longo dos anos.

I would also like to thank Professor Helmut Kettenmann and his lab, in the

Research Department Cellular Neuroscience from the Max-Delbrück Centrum in Berlin,

Germany for welcoming me in Berlin during my training period and for providing me with

so much valuable knowledge.

Gostaria também de agradecer às Professoras Doutoras Ana Sebastião e Maria

José Diógenes pela imensa disponibilidade que demonstraram desde o primeiro dia da

colaboração que encetámos e pelo seu contributo científico para este trabalho.

À Liliana Bernardino quero endereçar um agradecimento sentido pela ajuda tão

preciosa que me prestou na implementação das culturas organotípicas de cortes de

hipocampo. A tua enorme disponibilidade e simpatia, bem como a fé que sempre tiveste

que tudo ia correr bem, fizeram-me acreditar que seria possível completar esta tarefa

hercúlea!

Ao Pedro Pereira quero agradecer a ajuda preciosa nos aspectos mais técnicos

do trabalho com os cortes e pela paciência e disponibilidade que sempre demonstrou.

À Adelaide tenho prometido um merecido agradecimento desde o primeiro dia

em que entrei no CPM! Foste tu quem me encorajou a enveredar pelo mundo da ciência

e foste também tu quem me deu a mão nas vezes em que me perdi pelo caminho… Por

todos os momentos em que dispensaste o teu tempo para me ajudares, pelo enorme

contributo intelectual e científico que deste a esta tese e pela forma como me fizeste

sentir acarinhada neste grupo, um enorme obrigado! Sem a tua ajuda tenho a certeza

que este trabalho não seria o mesmo!

À Sofia tenho que agradecer os ensinamentos que me transmitiu durante este

período e a forma atenciosa e carinhosa como me tratou desde o início, fazendo-me

sentir em casa e parte integrante desta equipa! As nossas conversas à hora do lanche

foram uma fonte de motivação e de força para continuar, bem como o teu exemplo de

perseverança apesar das dificuldades!

Um agradecimento muito especial à minha companheira de jornada, Rita.

Fizemos esta travessia quase de mãos dadas e estivemos presentes uma para a outra

nos teus e nos meus momentos bons e maus. A tua amizade foi um factor determinante

para conseguir chegar até aqui e só espero ter correspondido na mesma medida!

Mereces toda a felicidade do mundo e eu tenho a certeza que a encontrarás e que

alcançarás o sucesso tanto a nível profissional como pessoal.

Quero agradecer também à Andreia, colega e amiga deste laboratório que se

tornou a nossa casa ao longo destes anos. Muito obrigado pela tua boa-disposição e por

toda a ajuda que me prestaste. Só tenho pena que não estejas aqui neste momento

mas esperamos o teu regresso!

Não posso deixar de agradecer às meninas do nosso laboratório e futuras

doutoras: à Inês, com quem tanto me identifico quer a nível pessoal quer a nível

profissional, muito obrigado por me ouvires tantas vezes e por me encorajares quando

desanimei. Tenho a certeza que também tu alcançarás este momento e todo o sucesso

que mereces! À pequena Ema que tanto cresceu desde que cá chegou! És um doce de

pessoa, sempre pronta para ouvir e ajudar e tão interessada nos outros e no mundo que

a rodeia. Tens crescido tanto nos últimos tempos que vamos ter que deixar de te

chamar “Pequena”! À Filipa quero agradecer todo o apoio que me ofereceste, o que

deste e o que não deste uma vez que é característica tua estar sempre pronta para

ajudar em qualquer circunstância! Desejo-te as maiores felicidades no teu percurso

académico e pessoal.

Não posso deixar de fora a Cibelle, a nossa Belli, que me ajudou tanto com o seu

conhecimento infinito sobre cortes e imunohistoquímica! Gostei muito de partilhar

contigo o mesmo espaço e de desenvolvermos esta amizade!

Gostaria ainda de agradecer à Eduarda, que por cá passou e avançou para

outros voos, por todas as conversas inspiradoras que tivemos e que me fizeram ter mais

força para superar esta prova! Mesmo não estando cá, a tua ajuda foi preciosa.

Agradeço também à Catarina Osório, que fez parte dos trabalhos aqui apresentados e

que sempre se mostrou disponível para me acompanhar neste percurso.

Um agradecimento especial para a Inês Milagre e para a Maria João Nunes que

perderam tanto tempo com as minhas dúvidas e questões e que nunca hesitaram em

me ajudar quando eu precisei! Gostaria também de agradecer à Professora Elsa, que

sempre se preocupou tanto comigo e que ao longo deste caminho me foi dando alento e

motivação para continuar! Cheguei ao fim e estou viva e de saúde! ☺

Quero agradecer a todos os inquilinos deste pequeno T0 que partilhamos aqui

na cave! Os momentos de diversão e as conversas animadas contribuíram e muito para

fazer deste sítio um local de trabalho muito agradável!

Quero agradecer especialmente às minhas colegas de faculdade que também

seguiram o caminho da ciência, Maria João e Cati, e que tanto me influenciaram nesta

escolha!

Quero agradecer também aos meus queridos amigos Rita, Cláudia e Badalo,

João e Ju e a todos os restantes membros da nossa grande família! Muito obrigado por

estarem sempre atentos e dispostos a ajudar e por me encorajarem constantemente!

Continuo a achar o condomínio fechado uma ideia vencedora! ☺

Aos meus sogros Fernanda e Guedes e aos meus cunhados Carla e Zé obrigado

por me fazerem sentir parte desta família e pelo interesse que demonstraram no meu

trabalho.

Aos meus manos Ana e Nuno quero agradecer do fundo do coração todo o amor

e carinho que têm por mim e que me deu força neste caminho. Saber-vos por perto e

saber que acreditam em mim e nas minhas capacidades tem sido o meu alento, a minha

motivação e só espero que se possam orgulhar de mim neste momento!

Aos meus sobrinhos Jaime e Vasco, Gui, Miguel e Francisco quero agradecer

por trazerem tanta luz e sorrisos para a minha vida! São vocês que fazem a vida ter

mais cor!

À minha mãe, um agradecimento sentido a quem sempre acreditou em mim e me

ensinou a acreditar em mim mesma. És a pessoa mais corajosa que conheço à face da

terra e o teu exemplo de força e coragem para superar a prova mais difícil de todas

inspira-me a dar o meu melhor para superar as pequenas provas da vida. Obrigada por

tudo!

Um agradecimento do fundo do meu coração e da minha alma vai para uma das

pessoas mais importantes da minha vida: o meu Pai. Dedico-te esta tese porque sempre

me ensinaste a ser perseverante e a não desistir nunca e por isso prometi a mim

mesma que completaria esta tarefa e que seria em tua honra. Espero que, estejas onde

estiveres, possas ter orgulho em mim…

Por fim, um agradecimento muito especial ao homem da minha vida, o Tiago.

Muito obrigado por todos os momentos que passámos juntos e também por entenderes

as minhas ausências ao longo deste caminho. Nunca, nem por um segundo, duvidaste

da minha capacidade de empreender esta tarefa e foi isso que me impulsionou a

conclui-la! Adoro-te!

Contents

xvii

Contents

Abbreviations ............................................................................................................. xxi Abstract ...................................................................................................................... xxv Resumo .................................................................................................................... xxvii

Chapter I - General Introduction .................................................................................. 1

1. The brain – an integrated network of interactive neurons and glia .......................... 3

1.1. Neurons ............................................................................................................ 4

1.2. Oligodendrocytes .............................................................................................. 5

1.3. Astrocytes ......................................................................................................... 5

1.4. Microglia ........................................................................................................... 6

1.4.1. Heterogeneity of microglial phenotypes .................................................... 6

1.5. Extracellular matrix ........................................................................................... 8

2. Role of microglia in different brain development stages .......................................... 9

2.1. Embryonic period .............................................................................................. 9

2.2. Neonatal period .............................................................................................. 10

2.3. Microglia in the aging brain ............................................................................. 10

3. Functional roles of microglia – activation and overactivation ................................. 11

3.1. Surveillance functions and role in synaptic plasticity ...................................... 12

3.2. Role of microglia in innate and adaptive responses ....................................... 14

3.2.1. Phagocytosis ........................................................................................... 14

3.2.2. Production of mediators in neuroprotection and neurodegeneration ....... 15

4. Microglial reactivity and modulation by cell interplay ............................................. 19

4.1. Models for the evaluation of microglial reactivity ............................................ 19

4.2. Reciprocal reactivity modulation by interplay of microglia with neighboring cells

22

5. Involvement of microglia in the progression of neurological diseases ................... 24

5.1. Acute and chronic neurological diseases ....................................................... 24

5.2. Neonatal hyperbilirubinemia ........................................................................... 27

5.2.1. Molecular mechanisms of bilirubin-induced CNS injury .......................... 30

6. Global aims of the thesis ........................................................................................ 33

7. References ............................................................................................................. 34

Chapter II - Features of bilirubin-induced reactive microglia: from phagocytosis to

inflammation ................................................................................................................ 47

1. Introduction ............................................................................................................ 50

Contents

xviii

2. Material and Methods ............................................................................................ 52

2.1. Chemicals ....................................................................................................... 52

2.2. Primary culture of microglia ............................................................................ 53

2.3. Cell treatment ................................................................................................. 54

2.4. Measurement of cytokine release ................................................................... 54

2.5. Western blot assay ......................................................................................... 54

2.6. Detection of NF-κB activation ......................................................................... 55

2.7. Morphological Analysis ................................................................................... 55

2.8. Assessment of microglial phagocytic properties ............................................. 56

2.9. Gelatin zymography ........................................................................................ 56

2.10. Evaluation of microglial cell death .................................................................. 56

2.11. Statistical analysis .......................................................................................... 57

3. Results ................................................................................................................... 57

3.1. UCB triggers IL-1β, TNF-α and IL-6 secretion following different temporal

profiles ....................................................................................................................... 57

3.2. p38 and ERK1/2 phosphorylation is elicited by UCB in microglia at an early time

point 58

3.3. NF-κB signalling pathway is triggered in UCB-activated microglia ................. 60

3.4. UCB-induced NF-κB translocation depends on both ERK1/2 and p38 .......... 61

3.5. Microglia depict morphological changes upon UCB stimulation ..................... 63

3.6. UCB differently modulates microglial phagocytosis depending on exposure time

64

3.7. Release of active MMPs is enhanced upon UCB stimulation of microglia ..... 65

3.8. UCB-stimulated microglia evidence enhanced COX-2 expression................. 66

3.9. UCB reduces microglial viability leading to loss of membrane integrity and

increased caspase activity ........................................................................................ 67

4. Discussion ............................................................................................................. 69

5. References ............................................................................................................ 74

Chapter III - Dynamics of neuron-glia interplay upon exposure to unconjugated

bilirubin ........................................................................................................................ 79

1. Introduction ............................................................................................................ 82

2. Material and Methods ............................................................................................ 83

2.1. Chemicals ....................................................................................................... 83

2.2. Primary culture of microglia ............................................................................ 84

2.3. Primary culture of astrocytes .......................................................................... 84

Contents

xix

2.4. Primary neuronal cell cultures ........................................................................ 85

2.5. Neuron-microglia mixed cultures .................................................................... 85

2.6. Cell treatment ................................................................................................. 85

2.7. Measurement of cytokine release ................................................................... 86

2.8. Gelatin zymography ........................................................................................ 86

2.9. Assessment of microglial phagocytic properties ............................................. 86

2.10. Quantification of nitrite levels .......................................................................... 86

2.11. Evaluation of cell death ................................................................................... 87

2.12. Neurite Extension and Ramification ................................................................ 87

2.13. Statistical Analysis .......................................................................................... 88

3. Results ................................................................................................................... 88

3.1. Conditioned media from UCB-treated astrocytes and neurons modulate

microglial secretion of cytokines, as compared to UCB-activated microglia ............. 88

3.2. Conditioned media from UCB-treated astrocytes and neurons have opposing

effects on microglial MMP-9 activity, as compared to UCB-activated microglia ........ 90

3.3. Nitric oxide (NO) release by microglia is elicited by UCB and is higher by naïve

microglia exposed to conditioned medium from UCB-treated neurons ..................... 90

3.4. Loss of viability in UCB-stimulated microglia is less in naïve microglia exposed

to conditioned medium from UCB-treated astrocytes (ACM), but higher if treated with

medium from neurons incubated with UCB (NCM) ................................................... 92

3.5. Microglial phagocytic properties enhanced by UCB further increase by medium

collected from neurons exposed to UCB ................................................................... 93

3.6. UCB-induced neuronal network impairment is prevented by microglia

environment ............................................................................................................... 95

3.7. Neuronal cell death triggered by UCB is diminished in the presence of microglia

97

4. Discussion .............................................................................................................. 98

5. References ........................................................................................................... 101

Chapter IV - Unconjugated bilirubin neurotoxicity is modulated by microglia and

prevented by glycoursodeoxycholic acid and interleukin-10 ............................... 105

1. Introduction .......................................................................................................... 108

2. Material and Methods .......................................................................................... 110

2.1. Chemicals ..................................................................................................... 110

2.2. Organotypic-cultured hippocampal slices ..................................................... 110

2.3. Preparation of microglia-depleted organotypic-cultured hippocampal slices 111

2.4. Organotypic-cultured hippocampal slices treatment ..................................... 111

Contents

xx

2.5. Assessment of cell death in organotypic-cultured hippocampal slices ......... 112

2.6. Quantification of nitrite levels ........................................................................ 112

2.7. Primary neuronal cell cultures ...................................................................... 112

2.8. Cell treatment ............................................................................................... 113

2.9. Neurite extension and ramification ............................................................... 113

2.10. Measurement of glutamate ........................................................................... 113

2.11. Evaluation of cell death ................................................................................ 114

2.12. Western blot assay ....................................................................................... 114

2.13. Statistical Analysis ........................................................................................ 114

3. Results ................................................................................................................. 115

3.1. Microglia modulate UCB-induced glutamate release and NO production in

organotypic-cultured hippocampal slices ................................................................ 115

3.2. NMDA receptors and NO are implicated in UCB-induced neurite impairment and

in cell death ............................................................................................................. 116

3.3. UCB-elicited accumulation of extracellular glutamate is reduced by both

GUDCA and IL-10, but not abolished ...................................................................... 119

3.4. GUDCA or IL-10 pre-treatment counteracts impairment of neurite outgrowth and

ramification, as well as cell death in UCB-treated neurons ..................................... 119

3.5. UCB decreases the expression of pre-synaptic proteins and this event is

abrogated by GUDCA, but not by IL-10 .................................................................. 121

3.6. Hampering of UCB-induced NO and glutamate production, as well as cell death

by GUDCA was reproduced in hippocampal slices ................................................. 123

4. Discussion ........................................................................................................... 124

5. References .......................................................................................................... 129

Chapter V - Final considerations ............................................................................. 135

1. Concluding remarks and perspectives ................................................................. 137

2. References .......................................................................................................... 143

xxi

Abbreviations

ACM Astrocyte conditioned medium

AD Alzheimer’s disease

AGUDC Ácido glicoursodesoxicólico

ALS Amyotrophic lateral sclerosis

AMPA α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate

AP-1 Activator protein-1

ATP Adenosine triphosphate

Aβ Amyloid β

BBB Blood-brain barrier

BDNF Brain-derived neurotrophic factor

BIND Bilirubin-induced neurological dysfunction

BNC Bilirrubina não conjugada

CNS Central nervous system

COX-2 Cyclooxigenase-2

CR3 Complement receptor 3

CSF Colony stimulating factor

DIV Days in vitro

ECM Extracellular matrix

ERK1/2 Extracellular signal regulated kinases 1 and 2

GABA γ-aminobutyric acid

GDNF Glial cell-line derived neurotrophic factor

GFAP Glial fibrillary acidic protein

GLT-1 Glutamate transporter-1

GTP Guanosine triphosphate

GUDCA Glycoursodeoxycholic acid

HIV Human immunodeficiency virus

HSA Human serum albumin

Iba1 Ionized calcium-binding adaptor molecule 1

Ig Immunoglobulin

IKK Inhibitor of nuclear factor-κB kinase complex

IL Interleukin

IL-1R Interleukin-1 receptor

iNOS Inductible nitric oxide synthase

xxii

IRAK 4 IL-1R-associated kinase 4

IκB Inhibitor of nuclear factor-κB

JNK1/2 c-Jun N-terminal kinases 1 and 2

LDH Lactate dehydrogenase

L-NAME N-ω-nitro-L-arginine methyl ester

LPS Lipopolysaccharide

LTP Long term potentiation

Mac 1 Macrophage receptor 1

MAP-2 Microtubule associated protein-2

MAPK Mitogen-activated protein kinase

MCAO Middle cerebral artery occlusion

MEKK MAPK kinase kinase

mGluR Metabotropic glutamate receptor

MHC Major histocompatibility complex

MK-801 [(+)-5-methyl-10,11-dihydro-

5Hdibenzo[a,d]cyclohepten-5,10-imine maleate)]

MMP Matrix metalloproteinase

MRI Magnetic resonance imaging

Mrp1 Multidrug resistance associated protein 1

MS Multiple sclerosis

MyD88 Myeloid differentiation factor 88

NCM Neuron conditioned medium

NF-κB Nuclear factor-κB

NGF Nerve growth factor

NMDA N-methyl-D-aspartate

nNOS Neuronal nitric oxide synthase

NO Nitric oxide

NT Neurotrophin

OPC Oligodendrocyte precursor cell

OSC Organotypic slice culture

OX Orexin

PAMP Pathogen-associated molecular pattern

PARP Poly-ADP ribose polymerase

PD Parkinson’s disease

PGE2 Prostaglandin E2

RNS Reactive nitrosative species

xxiii

ROS Reactive oxygen species

SNAP-25 Synaptosomal-associated protein-25

STAT Signal transducer and activator of transcription

TACM Conditioned medium from UCB-treated astrocytes

TF Transcription factors

TGF-β Transforming growth factor-β

TLR Toll-like receptor

TNCM Conditioned medium from UCB-treated neurons

TNFR Tumour necrosis factor receptor

TNF-α Tumour necrosis factor-α

TRAF TNFR-associated factor

TREM-2 Triggering receptor expressed on myeloid cells-2

UCB Unconjugated bilirubin

UDCA Ursodeoxycholic acid

Abstract

xxv

Abstract

Microglia are active sensors in the brain that rapidly engage adequate functional

activity states in response to injury to restore homeostasis. During the neonatal period,

the brain is more vulnerable to several injury conditions such as the one induced by

hyperbilirubinemia, a common situation observed in the newborn, where excessive levels

of unconjugated bilirubin (UCB) can reach and damage the brain. Although UCB-induced

neuronal and astrocytic toxicity have already been approached, the role of microglia in

this condition remains unclear. Thus, this thesis intended to investigate microglial

reactivity to UCB and to characterize the intervention of other brain cells in the

modulation of their response.

Isolated microglial cells showed to acquire a phagocytic phenotype upon UCB

exposure that preceded the release of pro-inflammatory cytokines. This release showed

to involve activation of upstream signalling pathways such as mitogen-activated protein

kinases (MAPKs) and nuclear factor-κB (NF-κB). We next investigated whether and how

the microenvironment influenced microglial response to UCB. Our findings revealed that

soluble factors released by UCB-stimulated astrocytes refrained microglial activation

while neuron-microglia interaction, evaluated using conditioned media and mixed culture

models, signalled microglial clearance functions but also enhanced its inflammatory

potential, ultimately leading to microglia demise. Finally, we evaluated microglial

neuroprotective or neurotoxic effects in a cell-to-cell concerted action in response to

UCB. Microglia revealed to participate in glutamate homeostasis, and to induce the

release of this neurotransmitter and of nitric oxide (NO) in UCB-treated organotypic-

cultured hippocampal slices, molecules that showed to be key players in UCB-induced

neurotoxicity. Moreover, our results point to interleukin (IL)-10 and glycoursodeoxycholic

acid (GUDCA) as promising therapies in neonatal hyperbilirubinemia.

In conclusion, microglia displays a dual activation profile in response to UCB

stimulation which is tailored by the influence of neighbouring cells. Collectively, these

data contribute for the understanding of microglia’s role in hyperbilirubinemia and

reinforce their remarkable functional plasticity.

Keywords: Astrocytes; Hyperbilirubinemia; Inflammation; Intracellular signalling;

Microglia; Neurons; Neuron-glia dynamics; Phagocytosis.

Resumo

xxvii

Resumo

As células da micróglia são sensores activos de dano no cérebro e rapidamente

adquirem estados funcionais de activação adequados à restauração da homeostase.

Durante o período neonatal o cérebro está mais vulnerável a certas lesões tais como as

induzidas pela hiperbilirrubinémia, uma condição comum no recém-nascido, na qual

níveis excessivos de bilirrubina não conjugada (BNC) podem atingir e lesar o cérebro. A

toxicidade desencadeada pela BNC em neurónios e astrócitos já foi alvo de estudos

prévios, no entanto o papel da micróglia nesta condição ainda não está completamente

esclarecido. Assim, foi objectivo desta tese investigar a reactividade da micróglia à BNC

e caracterizar a intervenção das restantes células do cérebro na modulação da sua

resposta.

As células de micróglia isoladas reagem à exposição à BNC pela aquisição de

um fenótipo fagocítico que precede a libertação de citocinas pró-inflamatórias. Esta

libertação parece envolver a activação a montante de vias de sinalização tais como as

cinases proteicas activadas por mitogénios (mitogen-activated protein kinases, MAPKs)

e o factor nuclear-κB (nuclear factor-κB, NF-κB). Em seguida investigámos a influência

do micro-ambiente celular na resposta da micróglia desencadeada pela BNC.

Verificámos então que astrócitos expostos à BNC libertam factores solúveis que contêm

a activação da micróglia. Por outro lado, a interacção entre neurónios e micróglia,

avaliada através do modelo dos meios condicionados e do das culturas mistas, parece

sinalizar a micróglia a empreender funções de eliminação de restos celulares, ao

mesmo tempo que aumenta o potencial inflamatório desta célula levando, em última

análise, à degeneração celular. Por fim, estudámos os efeitos neuroprotectores ou

neurotóxicos da micróglia face à BNC num modelo onde as interacções celulares são

preservadas. Os resultados obtidos em culturas organotípicas de fatias de hipocampo

revelam a participação das células da micróglia na homeostase do glutamato para além

de libertarem este neurotransmissor em conjunto com o óxido nítrico (nitric oxide, NO).

Os dados obtidos demonstram ainda que o glutamato e o NO são peças chave na

neurotoxicidade provocada pela BNC. Por fim, a interleucina-10 e o ácido

glicoursodesoxicólico (AGUDC) salientam-se como promissores agentes terapêuticos na

hiperbilirrubinémia neonatal.

Em conclusão, a micróglia apresenta um duplo perfil de activação em resposta à

BNC que é modulaado pela influência das células adjacentes. Em conjunto, estes dados

contribuem para uma melhor clarificação do papel da micróglia na hiperbilirrubinémia e

corroboram a sua notável plasticidade funcional.

Resumo

xxviii

Palavras chave: Astrócitos; Fagocitose; Hiperbilirrubinémia; Inflamação; Interacção

neurónio-glia; Micróglia; Neurónios.

1

Chapter I

GENERAL INTRODUCTION

General Introduction

3

1. The brain – an integrated network of interactive neurons and glia Neurons are the functioning unit of the central nervous system (CNS) and are

responsible for information processing. Glia make up 90% of the cells in the human

brain, and although their name derives from the greek word “glue”, research in the past

20 years has proven that these cells not only provide physical support to neurons but

make important contributions in the formation and operation of the neural circuitry and

play a key role in the brain’s immune functions (Fig.I.1) (Allen and Barres, 2009; Miller,

2005).

Fig. I.1. The brain as an integrated network. Glial cells (i.e. astrocytes, microglia and oligodendrocytes) are intimately related to neurons and blood vessels, contributing for their support and participating in various key brain functions. Adapted from Allen and Barres (2009).

The major difference between glia and neurons is that the latter ones fire action

potentials that underlie sensation, movement and thought, while glial cells lack this

capacity. Nevertheless, emerging research suggests that glial cells participate in

information processing and interact with synapses (Wake et al., 2009). In fact, the

Microglia

Astrocyte

Neuron

Oligodendrocyte

Bloodvessel

Chapter I

4

acknowledgement of neuron-glia crosstalk during synaptic transmission has

progressively challenged the classical view of the brain as a network of neuronal

contacts but rather as an integrated circuit of interactive neurons and glia (Bezzi and

Volterra, 2001). Hence glia is emerging as critical participants in every aspect of brain

development, function, and disease (Barres, 2008).

1.1. Neurons Neurons are highly specialized nerve cells responsible for communicating

information in both chemical and electrical forms throughout the body (Bloom, 2003).

Their structure comprises a cell body, or soma, from which an elaborate arborisation tree

emerges. Neuronal processes give neurons the regionalization of their functions, their

polarity and capacity to connect to other neurons, to sensory cells and to effector cells

(Hammond, 2001). Information is received and processed through the dendritic

branches, whose major structural components are the actin and microtubule

cytoskeleton together with microtubule-associated proteins (MAPs), responsible for

stabilization (Chen and Ghosh, 2005). The neuronal axon is a long cytoplasmic process

that culminates in a highly specialized structure, the pre-synaptic terminal, which,

together with the post-synaptic terminal of the adjacent neuron forms the synapse

(Bloom, 2003). The high molecular weight MAP-2 protein is more common in dendrites

than in axons and for this reason, MAP-2A and MAP-2B antibodies coupled to

fluorescent molecules are useful tools for labelling dendrites, allowing morphometric

analysis in cell cultures (Hammond, 2001).

Electrical signalling in the nervous system involves the movement of ions across

the neuronal plasma membrane through specific transmembrane proteins called ion

channels. These ionic currents evoke transient changes of membrane potential (action

potentials) which fire neuronal communication (Hammond, 2001). In the synapse,

neurotransmitters are released and establish communication between adjacent neurons

or between neurons and effector or sensory cells (Bloom, 2003). Several molecules

serve as neurotransmitters, such as acetylcholine, amino acids like glutamate, γ-

aminobutyric acid (GABA) and glycine and neuropeptides and their release at the pre-

synaptic terminal is induced by calcium-regulated vesicle exocytosis. Neurotransmitters

released into the synaptic cleft bind their specific receptors in the post-synaptic terminal

of the adjacent neuron and propagate action potential (Hammond, 2001).

Glutamate is the major excitatory neurotransmitter in the CNS and activates two

main types of post-synaptic receptors: ionotropic glutamate receptors that are ligand-

gated ion channels and are divided into N-methyl-D-aspartate (NMDA), α-amino-3-

hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) and kainate receptors, and


5

metabotropic glutamate receptors (mGluRs) that are receptors coupled to guanosine

triphosphate (GTP)-binding proteins. Glutamate’s concentration in the synaptic cleft must

be kept low as high or sustained glutamate receptor activation may induce neuronal

death via intracellular calcium and/or sodium deregulation, a mechanism called

excitotoxicity (Gras et al., 2006).

1.2. Oligodendrocytes Oligodendrocytes are the myelinating cells of the CNS and constitute about 5 to

10 % of the total glial population. Their processes enwrap neuron axons and form

myelin, an insulating lipid-rich membrane sheath that speeds the conduction of electrical

impulses. Therefore, saltatory propagation of action potentials occurs at the nodes of

Ranvier between oligodendrocyte myelin sheaths (Bradl and Lassmann, 2010).

Oligodendrocytes are vulnerable to cytotoxicity induced by dysfunctional astrocytes

(Sharma et al., 2010) or activated microglia as oligodendroglial cell death can be initiated

by increased levels of cytokines and reactive oxygen species (ROS) (Sherwin and Fern,

2005). Demyelination due to damage to oligodendrocytes leads to various diseases from

neurodevelopment to neurodegeneration such as periventricular leukomalacia (Volpe,

2009) and multiple sclerosis (MS) (Allen and Barres, 2009), respectively. Moreover, the

existence of direct chemical synapses between pyramidal neurons and oligodendrocyte

precursor cells (OPCs) in the mammalian hippocampus has been demonstrated,

underscoring the active role of glial cells in information processing (Bergles et al., 2000).

1.3. Astrocytes Astrocytes comprise about 85% of the glial population and are star-shaped cells.

They exert structural functions, are required for neuronal survival, neurite formation and

angiogenesis and maintain CNS homeostasis by regulating pH, ionic concentrations and

osmolarity (Montgomery, 1994). Astrocytes also provide metabolic support to neurons by

fueling their activity with energy and substrates and remove excess neurotransmitter

molecules from the extracellular space (Tsacopoulos and Magistretti, 1996). These cells

take part in the neurovascular unit of the blood-brain barrier (BBB) since their end-feet

ensheathe endothelial cells in micro-vessels (Cardoso et al., 2010). By signalling blood

vessels to expand or narrow, astrocytes regulate local blood flow to provide oxygen and

nutrients to neurons in need. Moreover, astrocytes were shown to induce tight junctions

in endothelial cells, thus participating actively in the establishment of the CNS

boundaries (Yamagata et al., 1997). More recently, bidirectional communication between

neurons and astrocytes was demonstrated. This fact altered the long-lasting paradigm of

the bipartite synapse, established between a pre- and a post-synaptic neuronal element,

Chapter I

6

leading to the novel concept of the tripartite synapse, where astrocytic participation in the

regulation of synaptic activity (Araque et al., 1999; Perea et al., 2009) and plasticity

(Barker and Ullian, 2010) is acknowledged. Moreover, astrocytes can communicate to

each other through the generation of calcium waves (Cornell-Bell et al., 1990) and to

neurons by the activation of calcium-based signalling cascades triggered by

neurotransmitters released by neurons which feedback the production of neuroactive

substances (Araque et al., 1999). Furthermore, Alvarez-Maubecin et al. (2000)

demonstrated gap-junctional electrical coupling between neurons and astrocytes and

also that selective changes in the membrane potential of glia modulate neuronal

excitability.

Considering these interesting concepts, a new role has been established for glial

cells, particularly astrocytes, in the so-called neuron-exclusive functions such as

information processing.

1.4. Microglia Microglia are small glial cells that reside within the CNS parenchyma and

constitute about 10 to 20 % of the total glial cell population (Chew et al., 2006; Vilhardt,

2005). They are ubiquitously distributed in the brain, being present in both grey and

white matter and exhibiting a higher density within the hippocampus, basal ganglia and

substantia nigra (Walter and Neumann, 2009).

Microglial cells share many properties of macrophages as they belong to the

mononuclear phagocyte lineage (Vilhardt, 2005). The notion that microglial cells are the

resident immune cells of the CNS has altered the concept of the brain as an

immunologically privileged organ supported by the existence of the BBB. Although

microglial immunocompetent functions are not on the same scale as that of peripheral

leucocytes, it is well established that they act as the brain’s innate immune system

(Aloisi, 2001; Streit, 2002).

1.4.1. Heterogeneity of microglial phenotypes The traditional classification of microglial subtypes described a resting and an

activated state. In the resting state microglia would display a ramified morphology, with

small bipolar or rod-shaped cell bodies bearing multiple branching processes (Chew et

al., 2006; Kim and de Vellis, 2005), while in the activated state, microglia would undergo

morphological changes acquiring an amoeboid appearance, up-regulate several cell

surface markers and produce a plethora of bioactive substances (Ladeby et al., 2005).


7

However, emerging research has updated the concept of microglial activation redefining

it as a shift in activity states rather than an activation process in itself (Fig. I.2).

Fig. I.2. Microglial activation is a shift between activity states. Microglia in its “resting” state plays a very important role in surveying the brain parenchyma (1) and can be prompted into an activated state (2) due to the presence of activating signals (such as inflammatory mediators and neurotransmitters) or the absence of constitutive calming signals, such as chemokines or neurotrophins that trigger a transition to an alerted state. During the activation process, microglia commit to distinct response phenotypes which can be altered throughout the pathological process (3, 4 and 4.1) by the influence of other nerve and immune cells (illustrated as feedback signals). At the end of this process, microglia can either return to a resting state (1), be eliminated (5) or step into a post-activated state (6), in which the cells acquire a kind of “memory” (indicated in the figure by a floppy disk icon). Adapted from Hanisch (2008).

Therefore, microglial activation should no longer be considered an all-or-none

event but an adaptive response to microenvironmental changes. (Hanisch and

Kettenmann, 2007). Indeed, Kreutzberg (1996) had already dubbed microglia as a

sensor of pathology.

The discoveries made by Nimmerjahn et al. (2005) and Davalos et al. (2005)

demonstrated that resting microglia showed highly motile extension and retraction of

processes which enabled them to constantly scan the neural parenchyma. These novel

1. Surveyingmicroglia

2. Alertedmicroglia

Activatingsignals

Calmingsignals

3. Reactive phenotype

4.1. Reactive phenotype

4. Reactive phenotype

Feedback signals

5. Elimination

6. Post‐activatedmicroglia

Chapter I

8

findings, that we will further explore below, produced a dramatic change in the concept of

resting microglia since this phenotype could no longer be considered dormant or inactive

but rather a surveying state with an important role in the normal and healthy brain

(Hanisch and Kettenmann, 2007; Raivich, 2005). Hence, microglia function as local

sentinels that can detect microdamages in the brain and rapidly repair such minute

insults without even being noticed. However, stronger or more prolonged insults may

trigger more drastic changes in microglial functional phenotype (Hanisch and

Kettenmann, 2007). Some authors now define activated microglia as a cell working to

restore CNS parenchymal homeostasis, in accordance with the new concept of

microglial activation phenotypes (Streit and Xue, 2009). The traditional classification of

microglial subtypes based on morphological criteria is no longer suitable, being replaced

by the definition of functional activation states (Schwartz et al., 2006). Furthermore,

microglia do not constitute a single and uniform cell population but comprise a family of

different cell phenotypes with ultimate beneficial or destructive outcomes, being

microglial responses tailored in regional and insult-specific manners (Carson et al.,

2007).

1.5. Extracellular matrix The extracellular matrix (ECM) accounts for about 20% of the adult brain volume

and is composed of collagen, proteoglycans, hyaluronan, fibronectin, elastins, laminins,

vitronectin, thrombospondins, tenascins, among other proteins and carbohydrates

(Zimmermann and Dours-Zimmermann, 2008). Mounting evidence demonstrates that

ECM is not merely mechanical scaffolding for nervous cells. ECM components are

involved in neural migration, proliferation, axon growth and guidance, or synapse

formation, processes that are essential during brain development. Furthermore, several

cell matrix receptors, like integrins, and cadherins have been shown to affect long-term

potentiation (LTP), revealing the implication of ECM in synaptic plasticity, as reviewed in

Pavlov et al. (2004). Interactions with ECM can also regulate immune cell action in both

a positive and negative fashion. For instance, ECM breakdown, from either microbe

colonization or resulting from the action of matrix metalloproteinases (MMPs) released

during inflammation, can produce fragments of specific ECM proteins that may act as

pro-inflammatory agents. In contrast, ECM proteins can also contribute to the down-

regulation of inflammation since decorin and biglycan were reported to inhibit the

classical pathway of complement activation (Morwood and Nicholson, 2006). Moreover,

the interaction of microglial receptors with ECM components such as cell adhesion

molecules contributes to microglial chemotaxis (Hristova et al., 2010; Raivich, 2005).


9

2. Role of microglia in different brain development stages The function of microglia depends on the age of the cell or organism (Walter and

Neumann, 2009). Therefore, we will next address the involvement of microglia in

different development stages of the brain (Fig. I.3).

Fig. I.3. Microglia along brain development. Summary of the role of microglia in different brain development stages, from embryonic period to the aging brain.

2.1. Embryonic period The origin of microglia has been a very contentious issue over the years.

However current view concerning microglial ontogenesis comprises a mesodermal origin

for microglia corroborating the original observations made by Pio del Rio-Hortega that

“microglia arise from polyblasts or embryonic cells of the meninges” (Cuadros and

Navascues, 1998). In fact, microglia are descendants of mesodermal fetal macrophages

which appear early in development and derive from primitive macrophages of the yolk

sac. The entry of fetal macrophages into the neuroepithelium and invasion through the

CNS occurs later in embryonic development, at about mid-gestation or earlier in rodents,

and late during the first trimester in humans (Alliot et al., 1999; Chan et al., 2007; Streit,

2001). Additionally, it is now accepted that microglial cells can be acutely derived from

blood-borne monocytes in the adult upon certain pathologic conditions (Graeber and

Streit, 2009; Kaur et al., 2001).

After their entry in the CNS, microglial precursors proliferate and spread through

the CNS by tangential and radial migration. Finally, mesodermal precursors originate

resident microglia that display an amoeboid-globoid appearance, with an enlarged cell

body and retracted processes (Chew et al., 2006; Cuadros and Navascues, 1998).

Microglia play an active role in CNS development and remodelling since they phagocyte

dead cells, cellular debris and aberrant axons (Kim and de Vellis, 2005; Streit, 2001).

Later in development microglia also participate in neuritogenesis, axonal growth and

• CNS developmentand remodelling

• Phagocytosis ofcellular debris

Embryonicdevelopment

• Residentimmune cellpopulation

• Reactivity to brain injury

Neonatal period

• Active pathologysensors

• Functionalplasticity

Adult stage

• Senescence anddystrophy

• Loss of function

Aging brain

Chapter I

10

guidance, as well as on the subsequent synaptogenesis and even in vasculogenesis.

The production of trophic factors by microglia also contributes for the development,

proliferation and growth of neurons and glia (Cuadros and Navascues, 1998; Nakajima

and Kohsaka, 2004).

2.2. Neonatal period In the neonatal period microglial cells transform into a more ramified morphology

constituting the resident immune cell population and presenting a slow turnover rate

(Cuadros and Navascues, 1998). An interesting study suggests that resident microglia,

in contrast to immune cells in other tissues, are not terminally differentiated along the

myeloid lineage. This immature state of microglial cells could explain their graded

response to activation as well as their enormous plasticity and capacity to assume

different phenotypes upon brain injury (Santambrogio et al., 2001).

Birth is not a deadline for the end of brain maturity since several key processes

such as synaptogenesis and gliogenesis take place postnatally rendering the brain highly

vulnerable to injury during the neonatal period. (Rice and Barone, 2000).

Interestingly, microglia has been implicated in neonatal pathologic conditions

such as hypoxic-ischemic (Vexler and Yenari, 2009) or excitotoxic brain injury

(Dommergues et al., 2003). In these cases, activated microglial cells rapidly accumulate

in the injured neonatal brain displaying up-regulation of cell surface markers such as

major histocompatibility complex I and II (MHC I and II) or complement receptors and

producing inflammatory mediators (Doverhag et al., 2010) and ROS. The inflammatory

response, in conjunction with excitotoxic and oxidative responses, is the major

contributor to ischemic injury in the immature brain (Vexler and Yenari, 2009).

2.3. Microglia in the aging brain An additional phenotype has recently been added to the concept of microglial

functional plasticity: a dystrophic or senescent phenotype (Streit et al., 2004). The

prevalence of degenerating microglia increases dramatically in Alzheimer’s disease

(AD), co-localizing with neurofibrillary degeneration (Graeber and Streit, 2009).

Moreover, fragmentation of cytoplasm (cytorrhexis) has been pointed to be indicative of

widespread microglial degeneration in amyotrophic lateral sclerosis (ALS) models

(Fendrick et al., 2007) and beading and clusters of fragmented twigs have also been

demonstrated in the aged brain (Hasegawa-Ishii et al., 2010). Such degenerative

changes may underlie a loss of microglial functionality and support, leading to

pathological outcomes like neurodegenerative changes and synapse loss (Streit and

Xue, 2009). This concept contributes for the growing notion that microglial activities are


11

essentially beneficial, becoming destructive only when they escape from the strict control

imposed on them (Schwartz et al., 2006).

3. Functional roles of microglia – activation and overactivation The definition of microglia’s function in the adult brain is not simple since it

encompasses the ability to respond to environmental changes and toxic insults (Fig. I.4).

Fig. I.4. Functional activation states of microglia. Resident microglia can become activated to adopt one of many diverse phenotypes depending on several circumstantial variables and these reactive phenotypes may be alternated along disease progression. Adapted from Perry et al. (2010).

Perhaps the most comprehensive term is “pathology sensor” coined by

Kreutzberg (1996). In fact, microglia assume an activated state and exhibit

immunological functions in response to danger signals. Nevertheless, several evidences

have demonstrated that overactivation might lead to microglial cell death, possibly as a

safety mechanism to prevent further deleterious effects (Liu et al., 2001; Polazzi and

Contestabile, 2006). In the following sections we will approach the several functions

performed by microglia in the healthy and injured brain.

fg

fg

hvSurveyingfunctions

Debrisclearance

Productionofinflammatorymediators

Neurotrophicfunctions

Chemotaxis–migration

Dystrophy–loss of function

Chapter I

12

3.1. Surveillance functions and role in synaptic plasticity As stated above, resident and “so-called resting” microglia are not functionally

silent cells. In vivo two-photon microscopy demonstrated that microglial processes are

remarkably motile presenting high velocities of extension and retraction interspersed with

brief static periods. By continuously sampling the environment with their highly motile

protrusions, microglia exert an important surveillance function and completely scan the

brain parenchyma every few hours (Nimmerjahn et al., 2005). Interestingly, adenosine

triphosphate (ATP) released from damaged tissue regulates microglial branch dynamics

in the intact brain and mediates a rapid microglial response towards injury (Davalos et

al., 2005).

Recently microglia have been shown to directly interact with synapses by

establishing intimate but transient connections with pre-synaptic and post-synaptic

elements. The frequency of these connections in the healthy brain corresponded with the

static periods observed by Nimmerjahn et al. (2005), while this connectivity increased

significantly during ischemic lesion (Wake et al., 2009) or after excitotoxic brain injury

(Hasegawa et al., 2007). Such observations led to the assumption that microglia monitor

the synapse functional status making brief contacts with the healthy ones and prolonged

contacts with the injured or pathological synapses. Additionally, pre-synaptic buttons

disappear after prolonged contact with microglia in ischemic conditions, suggesting that

microglia might produce synaptic stripping in a similar fashion as the one observed after

facial nerve axotomy (Wake et al., 2009). In this latter case, microglia become activated

and promote the detachment of afferent synaptic terminals from the surface of

motoneurons in an attempt to remove the excitatory input and limit further deleterious

outcomes (Kreutzberg, 1996; Moran and Graeber, 2004). These findings underscore

microglia’s function as sensors of the integrity of the CNS circuitry and may render them

a novel epithet as the CNS electrician (Graeber and Streit, 2009).

Moreover, mounting evidence of microglial interaction with synaptic elements

contributes for the concept that microglial cells also participate in synaptic plasticity, a

process connoted as a hallmark of neurons (Ben Achour and Pascual, 2010). Synaptic

plasticity is the basis of learning and memory and consists in a modification of synaptic

strength (Ben Achour and Pascual, 2010). Evidence of communication between

microglia, astrocytes and neurons relies on the expression of matching sets of

transmitters and receptors. Indeed, microglia express a variety of neurotransmitter

receptors such as AMPA, mGluRs, GABA receptor, as well as purinergic, cholinergic,

adrenergic, cannabinoid, dopaminergic and opioid receptors. Activation of those

receptors may trigger the release of several neuroactive substances like glutamate, ATP,

nitric oxide (NO), cytokines and chemokines (Pocock and Kettenmann, 2007).


13

The high density of purinergic P2X7 receptors in microglia mediates microglial

participation in the synapse and incited the emergence of quadpartite synapse concept

(Fig. I.5) (Bennett, 2007).

Fig. I.5. Microglia are vital members of the “quadpartite synapse”, which also includes pre-synaptic and post-synaptic neurons, and astrocytes. Input to the pre-synaptic terminal induces glutamate (Glu) release which binds to α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors on the post-synaptic neuron and also to AMPA receptors on the astrocytes end-feet, leading to the release of adenosine triphosphate (ATP) from the astrocyte. This acts as a chemoattractant driving microglia into intimate contact with synapses. ATP binds to P2X7 receptors on the pre-synaptic terminal, potentiating glutamate release from the terminal and acts on P2X7 receptors on microglia, leading to the release of tumour necrosis factor (TNF)-α which increases the effectiveness of the AMPA receptors on both the neuron and astrocytes. Adapted from Bennett et al. (2009).

ATP released from astrocyte processes at the synapse acts as a chemoattractant

on microglia driving them into intimate contact with synapses. Once microglia processes

are interlaced at the synapse they can themselves release ATP due to nerve terminal-

derived glutamate. ATP may also act on synaptic microglia P2X7 receptors to evoke the

release of cytokines such as tumour necrosis factor-α (TNF-α), which can then increase

AMPA receptors in the post-synaptic membrane processes. Thus the P2X7 receptor

ATP

P2X7

ATP

P2X7

TNF‐αAMPA

AMPA

NMDAGlu

Astrocyte Microglia

Pre‐synapticneuron

Post‐synapticneuron

Chapter I

14

mediates mechanisms that give rise to significant changes in the efficacy of synapses

(Bennett et al., 2009).

3.2. Role of microglia in innate and adaptive responses

Microglia constitute the first line of defence against invading microorganisms

(Town et al., 2005) and are intricately implicated in the initiation and propagation of the

inflammatory response (Chew et al., 2006). Activation of microglia can result in different

functional phenotypes entailing several characteristic features such as up-regulation of

cell surface markers, production of pro-inflammatory mediators and phagocytosis

(Hanisch and Kettenmann, 2007). Some authors refer to microglial activation as a

continuum where the innate or phagocytic response is at one end and the adaptive

response (comprising antigen presentation functions) is at the other (Town et al., 2005).

Therefore, the duration and type of stimulation received by microglia will determine the

activation features displayed by these cells reinforcing their remarkable plasticity in

response to injury (Town et al., 2005).

3.2.1. Phagocytosis Phagocytosis is defined as the process by which large particles (>0,5 µm) are

recognized, internalized and digested by phagocytic cells involving actin-dependent

mechanisms. This process is critical for the removal of infectious agents or senescent

cells and is performed by immune cells of the body such as macrophages and microglia.

Interaction of specific receptors expressed by phagocytic cells with ligands in the surface

of the particle is the first step for particle internalization, followed by actin polymerization

at the site of ingestion and internalization culminating in the formation of a

phagolysosome. Recognition mechanisms can be cell-mediated by cellular receptors like

integrins or mannose and scavenger receptors or humoral by the opsonisation of the

invading pathogen by antibodies or complement proteins which are then recognized by

specific receptors (Aderem and Underhill, 1999).

Activation of microglial phagocytic receptors can be associated with the

production of inflammatory products, as occurs in the case of Toll-like receptors (TLRs)

or Fc- receptors’ interaction with microbes (Neumann et al., 2009). On the other hand,

phagocytosis may occur in the absence of inflammation by the engagement of several

receptors such as complement, scavenger, phosphatidylserine (Neumann et al., 2009) or

purinergic (Koizumi et al., 2007) receptors as well as triggering receptor expressed on

myeloid cells-2 (TREM-2) (Takahashi et al., 2005). In fact, removal of apoptotic cells by

phagocytosis is described as a rather silent process which is essential during brain

development as well as in some pathologic conditions. Studies in hippocampal slices


15

show that microglia rapidly respond to neuronal death leading to phagocytic clearance

(Petersen and Dailey, 2004). Several putative “eat-me” signals have been established for

apoptotic cells such as phosphatidylserine externalization, interaction with vitronectin

receptors and changes in the pattern of glycosilation of surface proteins allowing

microglial recognition of apoptotic cells by lectin receptors (Ravichandran, 2003; Witting

et al., 2000).

As described previously, the phagocytic role of microglia in CNS development

and remodelling is widely established as these cells promote a clearance of dead or

dying cells and debris. In addition, microglial phagocytic activity is not limited to clearing

the corpses but it is also implicated in promoting axon pruning and cell death (Mallat et

al., 2005) as demonstrated by the studies of Marin-Teva et al. (2004) demonstrating that

microglia provoke the death of developing Purkinje cells by producing high levels of

superoxide. A putative hypothesis for the recognition of these Purkinje cells as prey for

phagocytic microglia involves the activation of caspase-3 which can lead to the

production of chemotactic agents like lysophosphatidylcholine (Lauber et al., 2003). A

role for microglia in the removal of unwanted synapses tagged by complement has also

been demonstrated (Stevens et al., 2007).

In the context of brain pathology, phagocytosis performed by microglia also plays

an essential role in the clearance of tissue debris and, therefore, in the generation of a

pro-regenerative environment (Neumann et al., 2009). In fact, microglia is able to

phagocyte myelin debris upon a demyelinating insult (Glezer et al., 2007; Takahashi et

al., 2007) and amyloid-β (Aβ) from senile plaques (Kakimura et al., 2002), thus triggering

repair. Nonetheless, an inadequate regenerative response may prevail if insufficient

clearance by microglia occurs (leading to pathology) or when microglial phagocytic

properties decline with aging (Zhao et al., 2006).

3.2.2. Production of mediators in neuroprotection and neurodegeneration Activated microglia release a plethora of soluble factors, which may be pro-

inflammatory and cytotoxic or, in contrast, exert neuroprotective or neurotrophic

functions (Block and Hong, 2005; Kim and de Vellis, 2005).

In fact, microglia are recognized producers of neurotrophic molecules such as

neurotrophins (NT), nerve growth factor (NGF), brain-derived neurotrophic factor

(BDNF), basic fibroblast growth factor, hepatocyte growth factor, glial cell-line derived

neurotrophic factor (GDNF), plasminogen, platelet-derived growth factor, among several

others (Nakajima et al., 2001a; Nakajima and Kohsaka, 2004). This panoply of

molecules have been reported to exert important roles in the development of the CNS

given their implication in the survival, maturation and differentiation of neurons,

Chapter I

16

astrocytes and oligodendrocytes as well as their involvement in neurite outgrowth and

synapse homeostasis (Kim and de Vellis, 2005; Nakajima and Kohsaka, 2004).

Furthermore, upon exposure to toxic insults, microglia also proved to secrete molecules

with a neuroprotective potential. For instance, lipopolysaccharide (LPS) stimulation of

microglia led to increased production of BDNF, NT-4/5 and NGF suggesting the

participation of these cells in neuronal regeneration (Miwa et al., 1997; Nakajima et al.,

2001a). After middle cerebral artery occlusion (MCAO), microglia secretes increased

amounts of insulin-like growth factor-1 that can promote neural precursors proliferation,

survival and differentiation, thus rendering microglia a pro-neurogenic role upon stroke

(Thored et al., 2009). In addition, glutamate may signal microglia to release neurotrophic

factors like BDNF, GDNF and NGF as an attempt to rescue neurons from excitotoxic

damage (Liang et al., 2010).

Microglia can also play additional neuroprotective actions as glutamate

scavengers by the expression of glutamate transporter-1 (GLT-1) (Nakajima et al.,

2001b). Moreover, GLT-1 expression by microglia is up-regulated upon motoneuron

injury in axotomized rat facial nucleus (Lopez-Redondo et al., 2000) or in response to

TNF-α production (Persson et al., 2005). Glutamine synthetase, the enzyme responsible

for converting glutamate into the less toxic glutamine, is also expressed in activated

microglia (Chretien et al., 2002; Rimaniol et al., 2000). Furthermore, the concept of

protective autoimmunity, developed by Schwartz et al. (2003) illustrates an interaction

between activated microglia and the adaptive immune system (namely T-cells) which

ultimately contributes for the orchestration of glutamate homeostasis (Schwartz et al.,

2003).

On the other hand, activation of microglia is also associated with the release of a

myriad of potentially cytotoxic substances such as ROS, proteases, pro-inflammatory

cytokines, chemokines, glutamate, among others (Aloisi, 2001; Kim and de Vellis, 2005).

In fact, microglia is one of the principal sources of cytokines in the brain, such as IL-1β,

TNF-α and IL-6 (Hanisch, 2002). Cytokines are low molecular weight proteins which

participate in a wide range of biological responses such as development and modulation

of inflammation (Acarin et al., 2001; Gebicke-Haerter, 2001). Interleukin (IL)-1β and TNF-

α are two important cytokines with pleiotropic functions (Fig. I.6). Both cytokines are

implicated in CNS inflammation given their ability to induce the expression of adhesion

molecules and chemokines leading to BBB breakdown and facilitating leukocyte

recruitment into the CNS (Allan and Rothwell, 2003; Hanisch, 2002; Lee and Benveniste,

1999).


17

Fig. I.6. Overview of the signaling pathways following cytokine receptor activation. Tumour necrosis factor (TNF)-α interacts with its specific receptor TNFR-1 initiating intracellular cascades through the recruitment of TNFR-associated factor (TRAF) 2. This event may lead to TNF-α-induced phosphorylation of members of the mitogen-activated protein kinases (MAPKs) family such as MAPK kinase kinases (MEKKs) and subsequently of the MAPKs [namely p38, extracellular signal regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinases 1 and 2 (JNK1/2)]. MAPKs activation may then induce the activation of transcription factors (TF) such as nuclear factor-kB (NF-kB). Stimulation of the NF-kB pathway may be initiated by the phosphorylation of the inhibitor of NF-κB (IκB) kinase complex (IKK) which is crucial for the degradation of IκB allowing NF-κB to translocate into the nucleus and promote gene transcription. Additionally, Interleukin (IL)-1 may also trigger NF-κB and MAPKs activation by the engagement of its membrane receptor IL-1R and consequent recruitment of myeloid differentiation factor 88 (MyD88). This latter adaptor protein associates with IL-1R-associated kinase 4 (IRAK 4) and subsequently activates TRAF6. These pathways may consequently result in modification of the inflammatory response and even cell death.

IL-1β has been broadly implicated in acute neuronal injuries such as the ones

resulting from ischemic or excitotoxic challenges (Hagberg et al., 1996). In fact, studies

where the action of IL-1β was inhibited culminated in marked reduction of the extent of

cell death elicited by ischemic, traumatic or excitotoxic brain injury (Allan and Rothwell,

Chapter I

18

2003). TNF-α was reported to promote neuronal (Venters et al., 2000) and

oligodendroglial cell death and is also involved in demyelination (Akassoglou et al.,

1998). Nevertheless, the neuroprotective or neurotoxic outcomes induced by TNF-α

seem to depend on the existence of two distinguishable signalling pathways mediated

through the TNF receptor (TNFR) 1 and 2 (Arnett et al., 2001). Indeed, a recent report

demonstrates that microglia up-regulates TNFR2 expression in response to an

inflammatory stimulus and that this event leads to the induction of anti-inflammatory

pathways (Veroni et al., 2010). In addition, IL-6 can have both anti- and pro-inflammatory

actions since it can significantly reduce ischemic brain damage after MCAO (Loddick et

al., 1998) or, in contrast, contribute to the pathophysiology of many diseases (Gadient

and Otten, 1997). Microglial cells express surface receptors for this multitude of

cytokines, thus propagating the inflammatory response (Aloisi, 2001). Among the

upstream signalling pathways involved in the production of cytokines by microglia are

mitogen-activated protein kinases (MAPKs), responsible for the phosphorylation of

several transcription factors such as nuclear factor-κB (NF-κB) or activator protein-1 (AP-

1) (Koj, 1996; Roux and Blenis, 2004). The nuclear translocation of these factors may

culminate in the production of the above described cytokines as well as chemokines,

complement factors and others (Nakajima et al., 2006).

A correlation between Notch-1 and NF-κB was recently confirmed in LPS-

stimulated microglia demonstrating that Notch signalling may amplify the pro-

inflammatory response of microglia by enhancing NF-κB signalling (Cao et al., 2010).

The signal transducers and activators of transcription (STAT) pathway might also be

activated in microglia upon ischemic lesion and lead to enhanced production of

inflammatory products (Justicia et al., 2000; Planas et al., 1997).

Cytokine release can also raise the production of other inflammatory mediators

such as NO (Chao et al., 1995). Microglial cells are reported to generate a burst of NO in

response to injury, which may lead to neuronal dysfunction and cell death (Bal-Price and

Brown, 2001; Cassina et al., 2002; Kawase et al., 1996; Sunico et al., 2010).

Interestingly, oxidative stress may lead to excessive glutamate production in microglia,

culminating in excitotoxic brain damage (Barger et al., 2007; Golde et al., 2002). The

release of glutamate in microglia is conveyed by the Xc antiporter, an heterodimeric

protein complex which exchanges extracellular cystine for intracellular glutamate (Barger

and Basile, 2001; Domercq et al., 2007). Glutamate is an excitatory neurotransmitter but

when present in the synaptic space above a certain threshold will lead to excitotoxicity,

that is, to the excessive activation of its receptors (namely NMDA, AMPA or kainate


19

receptors) with subsequent increase in intracellular calcium influx that culminates in

neuronal death (Johnston, 2005).

The extensive list of bioactive substances released by microglia in response to

brain injury encompasses molecules with chemoattractant properties such as

chemokines and MMPs (involved in the degradation of ECM components facilitating

leukocyte recruitment); molecules implicated in the initiation and propagation of

inflammation like prostanoids produced by cyclooxigenase-2 (COX-2) and complement

factors; substances involved in endoplasmic reticulum stress such as cathepsins; anti-

inflammatory cytokines like IL-10 and transforming growth factor (TGF)-β; and others

(Aloisi, 2001; Kim and de Vellis, 2005; Nakajima and Kohsaka, 2004).

The inflammatory response engaged by microglia has also been implied in the

inhibition of neurogenesis (Ekdahl et al., 2003; Monje et al., 2003) adding on the notion

that inflammation equals toxicity. Nevertheless, inflammation is a normal response of the

organism to infection, injury and trauma and some authors envisage this response as

primarily beneficial (Amor et al., 2010; Harry and Kraft, 2008) and as an internal

mechanism for repair (Schwartz et al., 2003). Yet, deregulation of the inflammatory

response and chronic immune activation can lead to deleterious effects and ultimately

cell death (Graeber and Streit, 2009; Harry and Kraft, 2008). It is, therefore, important to

recognize the beneficial aspects of the immune response and harness them, in order to

contribute to damage resolution instead of aggravating injury.

4. Microglial reactivity and modulation by cell interplay

4.1. Models for the evaluation of microglial reactivity Activation of microglia comprises the up-regulation of several surface markers,

which in the “resting” or “surveying” state are expressed at low or moderate levels (Aloisi,

2001; Guillemin and Brew, 2004). Some of these markers have been used to identify

microglia in tissue sections and cell cultures (Fig. I.7) but, the existence of a single

specific molecular marker for microglia is still unknown (Graeber and Streit, 2009). This

is explained by the fact that microglia share several antigens with macrophages,

endothelial cells, lymphocytes, among other cells (Guillemin and Brew, 2004). Therefore,

the known microglia markers are cell-specific in the sense that they do not label other

glial cells or neurons (Graeber and Streit, 2009). Some of the most commonly used

surface markers are lectins (carbohydrate binding proteins) such as wheat germ

agglutinin, mistletoe lectin, Ricinus communis agglutinin-1, Griffonia simplicifolia B4

isolectin and Lycopersicon esculentum (tomato) lectin, which present an increased

Chapter I

20

affinity for microglial cell but also bind neurons and myelin nodes and membranes

(Acarin et al., 1994). Other very commonly used markers are β2-integrins (CD11a,

CD11b and CD11c); the ligand for CD11a is intercellular adhesion molecule-1 whereas

the ligands for CD11b and CD11c are complement proteins (Kim and de Vellis, 2005).

CD11b is also commonly known as complement rerceptor-3 (CR3), and the antibodies

aimed at its recognition are orexin (OX) 42 or macrophage receptor 1 (Mac 1) depending

on the rat or mouse origin of the protein, respectively. Additional antibodies have been

proposed for the detection of activated microglia such as ED-1, also called CD-68, which

is expressed on the membranes of cytoplasmic granules such as phagolysosomes and

has been shown to correlate with phagocytic activity (Damoiseaux et al., 1994); or OX-6

that recognizes MHC class II antigens (Guillemin and Brew, 2004). One of the most

versatile immunocytochemical marker for microglia is ionized calcium-binding adaptor

molecule 1 (Iba1), an EF-hand protein which is up-regulated in activated microglia (Imai

et al., 1996).

Fig. I.7. Surface markers used for microglial labelling. Rat primary cortical microglia are labelled with ionized calcium-binding adaptor molecule 1 (Iba1) (A) or with orexin (OX) 42, an antibody raised against rat complement receptor 3 (B). Scale bar represents 20 µm.

However, the expression of these surface markers depends on the activation

state of microglial cells, as demonstrated by Cristovão and colleagues (2010), and its

presence is not always constant in all activation processes. For instance, activated

microglia accumulated in the facial nucleus after axotomy do not express ED-1 (Graeber

et al., 1998), and MHC class II can be also found in ramified microglia besides being

present in the activated state (Streit et al., 2004). These observations further corroborate

the functional plasticity exhibited by microglia. Additionally, different behaviors have been

A B


21

reported for microglial cells when applying in vitro or in vivo models which may be

attributable to the fact that these cells are always activated to same extent when in

culture somewhat limiting the ability to study microglial actual functions (Farber and

Kettenmann, 2005) and also to the lack of interaction with neighboring cells, a

determinant factor for the modulation of microglial reactivity (Hanisch and Kettenmann,

2007). The abundance of surface receptors expressed by microglia as well as the

panoply of soluble factors produced by these cells certainly vouches for its ability to

interact with other cells. The most striking example is the capacity of microglia to function

as an antigen presenting cell. Microglia bind the processed antigens to MHC complex on

its surface and engage interaction with T-cells. Optimal T-cell-microglia adhesion and

reciprocal activation are achieved through additional interactions between adhesion and

co-stimulatory molecules i.e. lymphocyte function-associated antigen 1, B7-1 and B7-2

molecules (CD80 and CD86), CD40 and their specific counterparts in the surface of T-

cells (Aloisi, 2001; van Kooten and Banchereau, 2000).

Therefore, when evaluating microglial response towards toxic or external stimulus

several experimental approaches can be used in order to explore the cross-talk between

microglia and nerve or immune cells (Fig. I.8). The conditioned medium model is used in

this thesis and aims at the evaluation of the influence of soluble factors on cell reactivity.

In fact, soluble factors released by either neurons or glial cells have proven to

reciprocally influence the development and reactivity of these cells (Gomes et al., 2001).

Mixed culture models, also applied in this thesis, rely on cell-to-cell interactions, such as

ligand-receptor interaction and phagocytosis. Finally, organotypic slice cultures (OSCs)

are heralded as the most approximate model to the in vivo conditions. In this model the

three dimensional architecture of the brain is maintained, recapitulating the cellular

contacts existent between neurons and glial cells. OSC are thick sections (250 – 400

µm) usually obtained from the early post-natal period by the roller tube method (in which

the cultured tissue is maintained attached to a coverslip rotating in the incubator) or by

the interface method developed by Stoppini et al. (1991), where explanted tissues are

layered onto a semiporous membrane and kept at an air – liquid interface (Lossi et al.,

2009).

Chapter I

22

Fig. I.8. Schematic representation of experimental models and respective main applications used for the evaluation of microglial reactivity.

4.2. Reciprocal reactivity modulation by interplay of microglia with neighboring cells

Neurons have been mainly regarded as the victims of overactivated microglia,

nevertheless, compelling evidence demonstrates the existence of reciprocal signalling

between neurons and microglia (Biber et al., 2007). Some authors have listed several

signals by which microglial function seems to be controlled and divided them in “off” or

“on” signals. “Off” signals are constitutively found in the healthy brain and its absence

may trigger microglial activation (Biber et al., 2007). The classical example of an “off”

signal is CD200, a member of the immunoglobulin (Ig) superfamily constitutively

expressed at the neuronal membrane, which is able to bind to its receptor CD200R, at

the surface of microglia, leading to the inhibition of microglial inflammatory functions

(Hoek et al., 2000). Among the “off” signals are also chemokines such as CX3CL1 or

fractalkine that exert a similar microglial inhibition (Mizuno et al., 2003; Zujovic et al.,

2000). Therefore, “off” signals can be envisaged as a way of neurons to maintain

microglia in a “resting” state in the healthy brain. “On” signals, by the other hand, are

described as substances produced by endangered or injured neurons and may lead to

microglial activation. Some of the “on” signals described are purines like ATP (Koizumi et

al., 2007), chemokines like CCL21 (Zhao et al., 2007) and MMPs (Kim et al., 2005),

CM

Isolatedcultures Conditionedmedium

Mixed cultures Organotypicslice cultures

Evaluation ofreactivity at a cellular andmolecular level

Assessment ofmodulatoryeffects ofreleasedsoluble factors

Investigationof cell‐to cellproximity –dependentinteractions

Evaluation ofcellularinterplay in a 3D brainstructure


23

which may induce microglial chemotaxis towards the injury site and enhance its

phagocytic activity. Whether the outcome resulting from neuron-microglia signalling is

either beneficial or detrimental is the question many researchers try to answer employing

different study models.

Using the conditioned medium model (Polazzi and Contestabile, 2006) found that

soluble factors released by healthy neurons lead to an overactivation of LPS-stimulated

microglia culminating in the apoptotic elimination of microglia, probably as a safety

mechanism. Reciprocally, activated microglia release factors such as NO that cause

neuronal cell death and neurite destruction (Munch et al., 2003). In addition, the severity

of neuronal injury appears to modulate the production of neurotrophic or neurotoxic

molecules by microglia (Lai and Todd, 2008). In fact, some studies have reported an

increased production of neurotrophins by microglia upon exposure to neuronal

conditioned medium (Nakajima et al., 2007). However, not only soluble factors but also

cellular interaction has to be accounted for when studying microglial response to

activating factors. In fact, if unstimulated microglia promotes neuronal survival by the

production of soluble factors, when in contact with neurons (in a mixed culture system)

and upon LPS stimulation, they lead to neuronal death, a situation that failed to occur

when conditioned media from LPS-stimulated microglia was applied to neurons (Zhang

and Fedoroff, 1996). Similarly, a proximity dependent mechanism, probably mediated by

NO, seems to govern microglial-induced neuronal death (Gibbons and Dragunow, 2006).

In contrast, there are also examples of neuron-microglia mixed cultures where neurons

seem to reduce microglial responsiveness to LPS (Chang et al., 2001). Microglia in

OSCs have demonstrated to migrate specifically towards sites of excitotoxic injury

(Heppner et al., 1998) and to participate in neuronal death in both excitotoxicity

(Bernardino et al., 2008; Dehghani et al., 2004) and hypoxia-ischemia models (Leonardo

et al., 2009) owing to the production of pro-inflammatory cytokines, NO and MMPs.

However, the neurotoxic or neuroprotective effects of microglia upon excitotoxic injury

may also depend on the concentration of inflammatory cytokines produced and on the

signalling pathways engaged (Bernardino et al., 2005).

Regarding microglial interplay with astrocytes, the latter cells produce substances

like colony stimulating factors (CSFs) which act as mitogens promoting microglial

proliferation (Suzumura et al., 1990). Moreover, microglia are able to sense astrocytic

calcium waves responding to it by the activation of purinergic receptors (Farber and

Kettenmann, 2005). Upon brain injury, nucleotides, chemokines and adhesion molecules

rapidly activated at the astrocytes’ surface act as chemoatractants driving microglia

towards the lesion site (Davalos et al., 2005; Raivich, 2005).

Chapter I

24

Conversely, astrocytes have been reported to down-regulate microglial ROS

production, probably as a mechanism of preventing excessive brain inflammation (Min et

al., 2006). The same was verified by Eskes et al. (2003) in an astrocyte-microglia mixed

culture where TNF-α release was decreased after exposure to a neurotoxicant. In

accordance, astrocytes were shown to counteract microglial glutamate-induced neuronal

death in a model using both conditioned media and mixed cultures (Liang et al., 2008). In

contrast, in a study employing OSCs, astrocytes react to excitotoxicity by the production

of the chemokines CXCL10 which interact with its specific microglial receptor culminating

in neuronal death (van Weering et al., 2010). Another study regarding the role of IL-18 in

neuropathic pain demonstrated that this cytokine mediates astrocyte-microglia

interaction and further enhances pain (Miyoshi et al., 2008).

Microglia has been long implicated in demyelinating diseases given its ability to

phagocyte myelin and to produce potential harmful factors such as TNF-α, which may

lead to oligodendroglial death (Selmaj and Raine, 1988a; Selmaj and Raine, 1988b). A

recent report showed that conditioned medium from LPS-activated microglia attenuated

primary OPC proliferation without inducing cell death (Taylor et al., 2010). In contrast,

microglial activation was shown to enhance OPC recruitment and to promote debris

removal, rendering the brain tissue more responsive to myelin repair (Glezer et al.,

2007).

In summary, interplay between CNS cells may have a strong and diverse

influence on the cascade of microglial responses and, in exchange, microglial activation

may also interfere with the reactivity of the remaining nerve cells leading to beneficial or

detrimental outcomes depending on the environmental circumstances.

5. Involvement of microglia in the progression of neurological diseases Microglial activation is the consequence of virtually all conditions associated with

neuronal injury. Therefore, the involvement of microglia in several neurological diseases

is notorious. In fact, the presence of activated microglia in regions of brain injury led to

the assumption that these cells where accountable for pathological effects (Harry and

Kraft, 2008). Nevertheless, emerging research as abovementioned proved the essential

roles of microglia in the brain’s normal functions as well as their neuroprotective effects

in some pathologic conditions.

5.1. Acute and chronic neurological diseases Among the most common acute neurological diseases are CNS infections by

different strains of bacteria, viruses or parasites (Rock et al., 2004). Innate immune cells

such as microglia interact with invading pathogens by pattern recognition receptors that


25

bind to structures designated as pathogen-associated molecular patterns (PAMPs)

(Janeway, 2001). Among the pattern recognition receptors are TLRs, CD14 receptor,

mannose receptors and CR3 and their interaction with PAMPs leads to the expression of

MHC class II and co-stimulatory molecules thus facilitating antigen presentation

(Janeway, 2001; Town et al., 2005). In fact, some authors stated that microglial role in

the defence of CNS against invading microorganisms relies mainly on their ability to “call

in the troops”, meaning systemic lymphocytes, monocytes and neutrophils (Rock et al.,

2004). Moreover, upon activation due to pathogen recognition, phagocytosis is engaged.

Phagocytic engulfment of the pathogen is often followed by a “respiratory burst” involving

the production of ROS (Block and Hong, 2005) aiming at eliminating the pathogen.

Microglia also participate in the opsonization of pathogens aiding in their recognition and

elimination (Hadas et al., 2010).

In acute CNS injury like entorhinal cortex injury (Rappert et al., 2004) or facial

nerve axotomy (Graeber et al., 1998) microglia rapidly migrates towards the lesion and

proceeds with the removal of injured neurons, thus exerting a pro-regenerative function.

In hypoxic-ischemic brain injury, a leading cause of mortality and morbidity, microglia

and macrophages accumulate at the lesion site (McRae et al., 1995) and may cause

injury through the production of pro-inflammatory mediators (Doverhag et al., 2010;

Hagberg et al., 1996) and consequent neuronal death (Dean et al., 2010). Yet,

contrasting data suggest that inhibition of microglial activation worsens hypoxic-ischemic

brain injury in a neonatal mouse model (Tsuji et al., 2004). In addition, ablation of

microglial cells results in significant increase in infarct size after MCAO in the adult stage

(Lalancette-Hebert et al., 2007). In this context microglia have also been demonstrated

to produce neurotrophic factors in response to stroke in the adult (Thored et al., 2009),

and therefore to promote recovery.

Compelling evidences have demonstrated that microglial activation is one of the

hallmarks of chronic neurodegenerative diseases such as AD, Parkinson’s disease (PD),

MS, ALS, human immunodeficiency virus (HIV) – associated dementia, among others

(Gebicke-Haerter, 2001), displaying both causative and exacerbating roles. The role of

microglia in neurodegenerative diseases is supported by the prevalence of progressive

neuronal loss and pathological levels of cytotoxic products like ROS and pro-

inflammatory cytokines, to the production of which microglia may be accounted for (Lull

and Block, 2010).

AD is a neurodegenerative disease characterized by the presence of insoluble

plaques containing Aβ and intraneuronal neurofibrillary tangles in the cortical region of

the brain (Lull and Block, 2010). The presence of MHC class II positive microglia was

Chapter I

26

reported in clusters formed around Aβ containing plaques in postmortem AD tissue

(McGeer et al., 1988; McGeer et al., 1987). Aβ recruits and activates microglia leading to

the release of NO (Ii et al., 1996), TNF-α (Dheen et al., 2005) and superoxide (Qin et al.,

2002), which may ultimately culminate in neuronal damage. Moreover, chemokine

receptors such as CX3CR1, which are critical for microglia – neuron communication

seem to be required for neuron loss in a mouse model of AD (Fuhrmann et al., 2010).

Although these evidences point to microglia as neurotoxic and disease aggravating in

AD, microglia was also reported to phagocyte Aβ (Das et al., 2003; Koenigsknecht-

Talboo and Landreth, 2005), thus playing a neuroprotective role. The clearance of

perivascular Aβ deposits by microglia seems to be mediated by the chemokine CCR2 (El

Khoury et al., 2007). In fact, immunization of animals with Aβ peptide proved to provide

protection against AD progression since it prompted microglial clearance of Aβ (Schenk

et al., 1999). However, clinical trials in humans were arrested due to the emergence of

meningoencephalitis symptoms (Senior, 2002). A contrasting theory defends that

microglia do not cause bystander damage but instead become senescent and

dysfunctional with normal aging, becoming disabled and deprived of their normal neuron-

supporting functions. Consistently, signs of microglial degeneration were found in AD

tissue, where microglia presented structural abnormalities and cell death (Streit, 2002).

PD is characterized by the degeneration of dopaminergic neurons in the

substantia nigra of the brain (Kim and Joh, 2006). Activated microglia was observed in

the striatum of postmortem PD brains (McGeer et al., 1988) and was associated with

neuronal damage due to the production of oxidative stress (Dexter et al., 1994) and

inflammatory mediators (Mogi et al., 1994). In addition, minocycline, a known blocker of

microglia activation, prevents loss of dopaminergic neurons (Wu et al., 2002), further

reinforcing the implication of inflammation and microglia in PD progression. However,

minocycline has also direct anti-apoptotic effects which may modulate microglial

response since this depends on the severity of cell death or injury (Kraft et al., 2009). In

fact, microglia may be neuroprotective in the early stages of disease but become

neurotoxic upon dopaminergic neuron degeneration (Sawada et al., 2006).

MS is the most prevalent inflammation-mediated demyelinating disease in which

the myelin sheath is damaged by an autoimmune response with a clear inflammatory

component (Block and Hong, 2005). Increased microglial activity was demonstrated

around MS lesions (Banati et al., 2000). Microglia may contribute for the disease

progression by the production of inflammatory cytokines and ROS (Murphy et al., 2010),

and also by their antigen-presentation function which sparks the autoimmune response

targeting myelin (Mack et al., 2003). Nevertheless, phagocytic clearance of debris has

been proven favorable for myelin repair (Glezer et al., 2007). In fact, a beneficial role has


27

been described for the microglial phagocytic TREM-2 receptor in an experimental

autoimmune encephalomyelitis (EAE) model (Piccio et al., 2007).

ALS is a very incapacitating neurodegenerative disorder characterized by the

degeneration of motor neurons (Henkel et al., 2009). Studies using a transgenic animal

model of ALS demonstrated the presence of structural abnormalities like cytorrhexis in

microglial cells (Fendrick et al., 2007), thus reinforcing the concept that microglial

dysfunction may underlie the onset or progression of chronic diseases.

HIV-associated dementia is a neurological syndrome that develops in the later

stages of HIV infection (Block and Hong, 2005). Microglia share some surface receptors

like CD4 and chemokine receptors with peripheral lymphocytes, which enables them to

harbor HIV and consequently act as viral replication sites (Garden, 2002). Indeed,

microglia are activated by HIV proteins such as Tat (D'Aversa et al., 2004) and gp120

(Kong et al., 1996) leading to the production of ROS, cytokines, chemokines and NO

(Brabers and Nottet, 2006) which ultimately lead to neuronal death. Some authors claim

even that persistent infection with HIV may disable or deplete microglia leading the way

for opportunist CNS infections and dementia (Streit, 2002).

5.2. Neonatal hyperbilirubinemia Hyperbilirubinemia is a frequent condition in the neonatal period, defined by an

increase in serum bilirubin above 5 to 7 mg/dl and occurring in up to 60% of full term

newborns and 80% of preterm infants (Porter and Dennis, 2002; Stevenson et al., 2001).

Generally designated as neonatal jaundice, it is characterized by a yellowish

discoloration of the skin and mucous membranes (Cohen et al., 2010; Porter and

Dennis, 2002). Unconjugated bilirubin (UCB) is formed by the catabolism of heme

(Gourley, 1997; Stevenson et al., 2001) and may reach excessive levels in newborn and

preterm infants due to its overproduction and to the limited ability to excrete this

molecule as a result of the of enzymatic machinery immaturity in these infants (Dennery

et al., 2001). In most cases, unconjugated hyperbilirubinemia occurs in a transient and

non-injurious manner and is called “physiologic jaundice” (Ostrow et al., 2002). When

UCB exceeds physiological concentrations, multifocal deposition of UCB may take place

in selected regions of the brain originating bilirubin encephalopathy (Gourley, 1997;

Ostrow et al., 2004). However, the serum bilirubin levels associated to the term

“physiologic jaundice” are under scrutiny since they may not accurately define a

harmless hyperbilirubinemia condition (Maisels, 2006). In fact, some studies have

reported a protective effect of UCB against oxidative stress injury, when present in low

levels (Dore and Snyder, 1999; Dore et al., 1999). Contrastingly, the same UCB levels

may reveal safe in normal newborn infants while become potentially hazardous in

Chapter I

28

premature infants (Stevenson et al., 2001; Watchko and Maisels, 2003) or even in term

neonates displaying additional pathologies, such as sepsis (Hansen et al., 1993; Kaplan

and Hammerman, 2005) or hypoxia-ischemia (Ahdab-Barmada and Moossy, 1984;

Falcão et al., 2007b), which may pose as risk factors for bilirubin encephalopathy. For

instance, data from magnetic resonance imaging (MRI) showed that severe cerebral

palsy occurred for relatively low serum bilirubin levels in preterm infants but only at high

levels in full terms (Gkoltsiou et al., 2008). Regarding prematurity as a risk factor for

bilirubin toxicity, data from in vitro (Falcão et al., 2005; Falcão et al., 2006) and in vivo

studies (Conlee and Shapiro, 1997; Keino and Kashiwamata, 1989) point to the

existence of windows of developmental susceptibility of the CNS to bilirubin toxicity

(Shapiro, 2010).

The term acute bilirubin encephalopathy is used to describe the sharp

manifestations of bilirubin toxicity seen in the first weeks after birth (American Academy

of Pediatrics, 2004). Such manifestations, that might be reversible, progress from

lethargy and decreased feeding to hypotonia and hypertonia, high-pitched cry,

impairment of upward gaze, fever and seizures (Shapiro, 2003). Recommended

therapeutic approaches for acute bilirubin encephalopathy are phototerapy and in the

more severe cases exchange transfusion (American Academy of Pediatrics, 2004).

The classic form of chronic bilirubin encephalopathy is called kernicterus,

originally a pathological term referring to the yellow staining (-icterus) of the deep nuclei

of the brain (kern-, relating to the basal ganglia) (Shapiro, 2010). This condition is

characterized by chronic and permanent sequelae like athetoid cerebral palsy, deafness

or hearing loss, impairment of upward gaze and dental enamel hypoplasia, or even

death. Brain regions more prone to neuropathological lesions produced by

hyperbilirubinemia are the globus pallidus and subthalamic nucleus, auditory and

oculomotor brainstem nuclei, cerebellum and hippocampus (Shapiro, 2003; Shapiro,

2005). The increasing prevalence of bilirubin encephalopathy (Ebbesen, 2000; Hansen,

2000) due to earlier hospital discharge and to the implementation of less aggressive

clinical approaches in the management of neonatal jaundice (Maisels and Newman,

1998) has reawakened interest in understanding the pathophysiology of this condition.

Moreover, the incidence of severe jaundice is at near epidemic proportions among some

developing countries (Gordon et al., 2005; Katar, 2007; Owa and Ogunlesi, 2009).

There is evidence that even moderate levels of unconjugated bilirubin can

produce subtle encephalopathy, referred to as bilirubin-induced neurological dysfunction

(BIND) (Shapiro, 2003; Shapiro, 2005). BIND is associated with cognitive disturbances,

mild neurological abnormalities (Hyman et al., 1969; Odell et al., 1970; Rubin et al.,

1979), isolated hearing loss (Bergman et al., 1985; Salamy et al., 1989) and auditory


29

neuropathy (Rance et al., 1999; Simmons and Beauchaine, 2000). Recent data have

demonstrated a high incidence of acute auditory neuropathy spectrum disorder among

neonates with severe jaundice (Saluja et al., 2010). Nonetheless, auditory dysfunction

may occur in children with or without other signs of classical kernicterus given the high

sensitivity of the auditory system to bilirubin toxicity (Shapiro and Nakamura, 2001). The

cellular mechanism underlying bilirubin-induced hearing dysfunction seems to involve

protein kinases A and C – mediated GABA/glycinergic synaptic transmission in postnatal

rat ventral cochlear nucleus neurons (Li et al., 2010). In addition, hyperbilirubinemia has

been shown to cause degeneration of excitatory synaptic terminals in the auditory

brainstem and this is associated with activation of neuronal nitric oxide synthase (nNOS)

(Haustein et al., 2010).

Regarding cognitive disturbances and neurological abnormalities, moderate

hyperbilirubinemia has been associated with minor neurologic dysfunction throughout the

first year of life (Soorani-Lunsing et al., 2001). Additional studies have demonstrated that

neonatal hyperbilirubinemia can have an impact on learning and memory later in infancy

(Zhang et al., 2003) as well as affect long-term cognitive ability (Seidman et al., 1991).

An interesting association has also been made between hyperbilirubinemia and

increased vulnerability for later development of mental disorders (Dalman and Cullberg,

1999). In fact, neonatal jaundice has been considered a perinatal risk factor for autism

spectrum disorders and sensory processing disorder given its higher prevalence in

children suffering from these disorders (May-Benson et al., 2009). An association

between hyperbilirubinemia and developmental delay, attention-deficit disorder, and

autism was also observed (Jangaard et al., 2008). Similarly, schizophrenic patients

showed a significantly higher incidence of hyperbilirubinemia relative to patients suffering

from other psychiatric disorders, and the psychiatric symptom score was significantly

higher in schizophrenic patients with hyperbilirubinemia than in patients without

hyperbilirubinemia (Miyaoka et al., 2000). Another study using Gunn rats, the animal

model for bilirubin encephalopathy, demonstrated that these animals displayed

stereotypical behaviors, considered as positive symptoms of schizophrenia, as well as

more aggressive behaviors (Hayashida et al., 2009). These findings further strengthen

the neurodevelopmental hypothesis which proposes that a proportion of schizophrenia is

the result of an early brain insult, either pre or perinatal, which affects brain development

leading to abnormalities expressed in the mature brain (Gupta and Kulhara, 2010). In

fact, UCB has been proven to cause impairment of neurotransmitter release in synaptic

vesicle membranes (Roseth et al., 1998), alteration of long-term synaptic plasticity

(Chang et al., 2009) and decreased synaptic activity in hippocampal slices (Hansen,

1994). In addition, UCB leads to neurite outgrowth impairment in immature cortical

Chapter I

30

neurons (Falcão et al., 2007c) and also interferes with axonal growth cone area and

spine formation (Fernandes et al., 2009). These findings further confirm that UCB’s

effects in the developing brain may lead to decreased synaptic connectivity, which may

underlie the emergence of neurodegenerative diseases.

5.2.1. Molecular mechanisms of bilirubin-induced CNS injury Bilirubin-induced neurotoxicity is initiated at the level of the membrane. Indeed,

UCB interacts with whole nerve cell and mitochondrial membranes disrupting its redox

status and increasing oxidative damage (Rodrigues et al., 2002b). Rough endoplasmic

reticulum abnormalities were also observed in astrocytes exposed to UCB (Silva et al.,

2001a). UCB-induced membrane permeabilization leads to mitochondrial swelling and

release of cytochrome c (Rodrigues et al., 2000b), which is followed by caspase-3

processing, poly-ADP ribose polymerase (PARP) cleavage and Bax translocation,

resulting in apoptotic cell death (Rodrigues et al., 2002a). Studies have also showed the

involvement of caspase-8 in cell death provoked by UCB (Seubert et al., 2002).

Susceptibility to UCB-induced damage differs with age and cellular type. Younger cells

are more susceptible to UCB-induced injury (Falcão et al., 2005; Falcão et al., 2006;

Rodrigues et al., 2002c; Silva et al., 2002) and to this may account the reduced

expression of multidrug resistance associated protein 1 (Mrp1) in immature cells (Falcão

et al., 2007a), an efflux pump involved in UCB export. Moreover, neurons are more

vulnerable to death mechanisms than astrocytes (Falcão et al., 2006; Silva et al., 2002),

in which an inflammatory response is triggered by UCB (Fernandes et al., 2004).

Neurons are also more exposed than astrocytes to oxidative injury by UCB, for which

accounts the lower glutathione stores in neuronal cells (Brito et al., 2008b) (Fig. I.9).

In addition, UCB causes a bioenergetic and oxidative crisis in immature neurons,

inhibiting cytochrome c oxidase activity and increasing glycolitic activity as well as ROS

production (Vaz et al., 2010). Interestingly, a recent report demonstrated that oxidative

damage by UCB may be mediated by overstimulation of glutamate receptors (Brito et al.,

2010).

Glutamate and NMDA receptors are particularly important in the pathogenesis of

neonatal neuronal injury (Johnston, 2005). In fact, UCB inhibits glutamate uptake by

astrocytes (Silva et al., 1999) prolonging glutamate’s presence in the synaptic cleft,

which may engender NMDA receptors overstimulation and excitotoxicity in both in vitro

(Grojean et al., 2000; Grojean et al., 2001) and in vivo studies (Hoffman et al., 1996;

McDonald et al., 1998).

Astrocytes and microglia react to UCB through the increased production of pro-

inflammatory cytokines and glutamate (Fernandes et al., 2006; Fernandes et al., 2004;


31

Gordo et al., 2006). Upstream signalling pathways involved in astrocytic cytokine

production elicited by UCB are MAPKs and NF-κB, which also contribute to the observed

cell death (Fernandes et al., 2007a; Fernandes et al., 2006). In addition, activation of p38

MAPK is involved in bilirubin-induced granule cerebellar neuronal death (Lin et al.,

2003). Moreover, decreased viability and apoptotic and/or necrotic cell death is also

observed in oligodendrocytes (Genc et al., 2003) and microglial cells exposed to UCB

(Gordo et al., 2006).

Fig. I.9. Major findings regarding the molecular mechanisms involved in unconjugated bilirubin (UCB)-induced toxicity. UCB triggers several toxicity-related molecular mechanisms in neurons, astrocytes and microglia. Moreover, the intensity of the observed deleterious effects depends on cell type and also on maturation stage, given that both immature neurons and astrocytes are more vulnerable to UCB-induced toxicity. (DIV – days in vitro).

Concerning potential therapeutical strategies to prevent UCB-induced injury,

glycoursodeoxycholic acid (GUDCA) and IL-10 have already been assayed with positive

results. Indeed, ursodeoxycholic acid (UDCA) and its conjugates have demonstrated

neuroprotective effects by the stabilization of mitochondrial membranes (Sola et al.,

2002), inhibition of mitochondrial swelling (Rodrigues et al., 2000c), cytochrome c

release (Rodrigues et al., 2000a) and, ultimately, by the decrease of apoptotic cell death

(Silva et al., 2001b). UDCA oral administration induces a rapid and sustained decrease

in plasma UCB concentrations in Gunn rats (Cuperus et al., 2009). Moreover, recent

reports have described GUDCA to prevent UCB-induced alterations in protein oxidation,

lipid peroxidation, impairment of glutathione homeostasis and neuron cell death (Brito et

al., 2008a) as well as its ability to ameliorate UCB-induced mitochondrial respiratory

chain dysfunction and to restore cellular antioxidant potential (Vaz et al., 2010). In

Microglia

Cell death (necrosisand apoptosis)

Cytokine releaseGlutamate secretion

Morphological changes

Astrocyte

Cell death (necrosisand apoptosis) Oxidative stressCytokine releaseGlutamate

secretion and reuptakeinhibition

Neuron

Cell death (necrosisand apoptosis)

Oxidative stressCytokine releaseGlutamate secretion

Sensitivityalong DIV

Sensitivityalong DIV

Chapter I

32

addition, both GUDCA and IL-10 can modulate astrocytic reactivity to UCB decreasing

the elicited inflammatory properties and reducing cell death (Fernandes et al., 2007b).


33

6. Global aims of the thesis The main goal of the present work is to explore the role of microglia in

hyperbilirubinemia by addressing microglial reactivity, characterizing the dynamics of

neuron-glia interplay and identifying potential therapeutic strategies.

As a first step we sought to investigate the cellular and molecular mechanisms of

isolated microglial reactivity towards UCB by the evaluation of cell survival and by the

assessment of the different phenotypes acquired by microglia when facing UCB.

Therefore we intended to evaluate microglial phagocytic abilities and to characterize the

inflammatory response engaged by these cells by studying the temporal profile of pro-

inflammatory cytokines’ secretion as well its underlying signalling pathways and by the

analysis of indicators of the inflammatory response such as MMPs activation and COX-2

up-regulation.

Since microglia exhibited different activation stages in the presence of UCB,

displaying both phagocytic and inflammatory phenotypes in a sequenced fashion along

exposure time, we decided to study how neuron-glia interplay could modulate the effect

of UCB. For this matter we used a conditioned media model to evaluate the influence of

soluble factors released by UCB-stimulated astrocytes and neurons in microglial

reactivity. We also assessed the influence of neuron-microglia cell-to-cell interactions in

UCB-induced deleterious effects on immature neurons in a neuron-microglia mixed

culture.

Finally, we advanced one step further and sought to investigate the role of

microglia in the modulation of UCB-induced neurotoxicity in organotypic-cultured

hippocampal slices where selective ablation of microglia was performed. Furthermore,

we aimed at unravelling the molecular mechanisms triggering UCB-induced neurotoxicity

which may underlie the association of hyperbilirubinemia with mental disorders.

Ultimately we evaluated the ability of GUDCA and IL-10 to prevent UCB’s deleterious

effects in both neuronal cultures and organotypic-cultured hippocampal slices.

Chapter I

34

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47

Chapter II

FEATURES OF BILIRUBIN-INDUCED REACTIVE MICROGLIA: FROM PHAGOCYTOSIS TO INFLAMMATION

Sandra L. Silva, Ana R. Vaz, Andreia Barateiro, Ana S. Falcão, Adelaide Fernandes, Maria A. Brito, Rui F. M. Silva, Dora Brites

Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL),

Faculdade de Farmácia, University of Lisbon,

Av. Professor Gama Pinto, Lisbon 1649-003, Portugal.

Neurobiology of Disease (2010) 40:663-675.

48

Acknowledgments

The authors thank Elsa Rodrigues for her expertise with gene reporter assays. This work

was supported by POCI/SAU-MMO/55955/2004 and PTDC/SAU-NEU/64385/2006

grants, from Fundação para a Ciência e a Tecnologia (FCT), Lisbon, Portugal and

FEDER (to D.B.). S.L.S. was recipient of a PhD fellowship (SFRH/BD/30326/2006) from

FCT.

Unconjugated bilirubin-induced phagocytic and inflammatory role of microglia

49

Abstract Microglia constitute the brain’s immunocompetent cells and are intricately

implicated in numerous inflammatory processes included in neonatal brain injury. In

addition, clearance of tissue debris by microglia is essential for tissue homeostasis and

may have a neuroprotective outcome. Since unconjugated bilirubin (UCB) has been

proven to induce astroglial immunological activation and neuronal cell death, we

addressed the question of whether microglia acquires a reactive phenotype when

challenged by UCB and intended to characterize this response.

In the present study we report that microglia primary cultures stimulated by UCB

react by the acquisition of a phagocytic phenotype that shifted into an inflammatory

response characterized by the secretion of the pro-inflammatory cytokines tumour

necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, up-regulation of cyclooxygenase

(COX)-2 and increased matrix metalloproteinase (MMP)-2 and -9 activities. Further

investigation upon upstream signalling pathways revealed that UCB led to the activation

of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB at an early

time point, suggesting that these pathways might underlie both the phagocytic and the

inflammatory phenotypes engaged by microglia.

Curiously, the phagocytic and inflammatory phenotypes in UCB-activated

microglia seem to alternate along time, indicating that microglia reacts towards UCB

insult firstly with a phagocytic response, in an attempt to constrain the lesion extent and

comprising a neuroprotective measure. Upon prolonged UCB exposure periods, either a

shift on global microglia reaction occurred or there could be two distinct sub-populations

of microglial cells, one directed at eliminating the damaged cells by phagocytosis, and

another that engaged a more delayed inflammatory response. In conclusion, microglial cells are relevant partners to consider during bilirubin

encephalopathy and the modulation of its activation might be a promising therapeutic

target.

Keywords: Microglial activation; Cyclooxygenase-2; Hyperbilirubinemia; Inflammatory

signalling pathways; Matrix metalloproteinases; Mitogen activated protein kinases;

Nuclear factor-κB; Phagocytic activity.

Chapter II

50

1. Introduction

Hyperbilirubinemia is a common condition in the neonatal period and results from

a limited ability of the newborns to excrete an over produced bilirubin (Dennery et al.,

2001; Watson, 2009). Physiological to pathological transition is driven by the multifocal

deposition of unconjugated bilirubin (UCB) in selected regions of the brain leading to

encephalopathy and kernicterus (Hansen, 2002; Porter and Dennis, 2002). This event is

directly correlated with death, as well as with impairments of neural development and

hearing (Oh et al., 2003). Additionally, moderate hyperbilirubinemia has been proven to

be associated with a significant increase in minor neurologic dysfunction throughout the

first year of life (Soorani-Lunsing et al., 2001) and has also been related to the outcome

of mental disorders such as schizophrenia (Miyaoka et al., 2000).

The cytotoxic effects of UCB in the central nervous system (CNS) have been

broadly studied and comprise several features such as: perturbation of nerve cell and

mitochondria membranes (Rodrigues et al., 2002b; Rodrigues et al., 2002c); inhibition of

glutamate uptake prolonging its presence in the synaptic cleft (Silva et al., 1999; Silva et

al., 2002); N-methyl-D-aspartic acid (NMDA)-mediated excitotoxicity (Brito et al., 2010;

Grojean et al., 2000; Grojean et al., 2001; McDonald et al., 1998); and increase in

intracellular calcium (Brito et al., 2004). All these events may culminate in cell death by

both necrosis and apoptosis (Rodrigues et al., 2002a; Silva et al., 2001) being neurons

more susceptible to death mechanisms than astrocytes (Falcão et al., 2006; Silva et al.,

2002). Some of the injurious effects of UCB on astrocytes are an elevated glutamate

secretion and the activation of inflammatory pathways that lead to cytokine release

(Falcão et al., 2006; Fernandes et al., 2006; Fernandes et al., 2004). Furthermore, our

group was the first to demonstrate that UCB activates and damages microglial cells

(Gordo et al., 2006). Indeed microglia showed to be the most reactive brain cells when

compared to astrocytes and neurons, as they evidence increased UCB-induced cell

death, release of glutamate and cytokine production (Brites et al., 2009).

Microglial cells reside within the CNS parenchyma (Streit, 2002) and engage

several important roles in the developing brain (Cuadros and Navascues, 1998; Kim and

de Vellis, 2005) as well as in pathological conditions (Block and Hong, 2005; Nakajima

and Kohsaka, 2004). In response to injury, microglia turn into an activated state and

display a complexity of phenotypic alterations that illustrate what is called “reactive

microglia”. This activation entails several features such as: dramatic morphologic

changes by the acquisition of an amoeboid phenotype (Kreutzberg, 1996); up-regulation

of intracellular enzymes and cell surface markers, release of pro-inflammatory mediators,

oxygen radicals and proteases (Kim and de Vellis, 2005), antigen presentation (Aloisi,

2001) and phagocytosis (Chew et al., 2006).


51

Moreover, the implication of microglia to neonatal pathologic conditions has been

acknowledged (McRae et al., 1995; Vexler and Yenari, 2009), since the production of

inflammatory mediators by these cells is a major contributor to hypoxic- ischemic injury in

the neonatal brain (Doverhag et al., 2010).

Microglial activation must not be viewed as an “on/off” process, but rather as a

shift between activity states, altering between a surveying and an effector status. In fact,

microglia’s activation process is an adaptive one but, depending on the circumstances in

which it occurs, may have neuroprotective or neurotoxic outcomes (Hanisch and

Kettenmann, 2007). Indeed, microglial involvement in various neurodegenerative

disorders is notorious, namely in Parkinson’s disease (Tansey et al., 2008), Alzheimer’s

disease (Kim and Joh, 2006), multiple sclerosis (Jack et al., 2005; Muzio et al., 2007),

and human immunodeficiency virus (HIV)-associated dementia (Gonzalez-Scarano and

Baltuch, 1999), mostly owing to its inflammatory character. Yet, microglial phagocytic

role in numerous neurodegenerative diseases as well as in acute brain injury is essential

for tissue debris removal and contributes for a pro-regenerative environment (Neumann

et al., 2009). Moreover, phagocytic clearance of debris may be considered a protective

measure as it constitutes an attempt to restrain further detrimental inflammatory

responses (Napoli and Neumann, 2009).

In this study we characterize the microglial response to UCB stimulation by

evaluating both its phagocytic properties and the inflammatory mechanisms engaged

upon activation. We observed, for the first time, that UCB induces an increase in the

phagocytic properties of microglia, followed by a shift into a rather inflammatory

response with prolonged exposure time. Moreover, this inflammatory response triggered

by UCB follows different temporal profiles of interleukin (IL)-1β, tumour necrosis factor

(TNF)-α and IL-6 secretion. Remarkably, an increase in TNF-α and IL-1β release is

observed prior to the secretion of IL-6. Our findings also point to mitogen-activated

protein kinases (MAPKs) and nuclear factor (NF)-κB as probable signalling pathways

involved in microglial reactivity to UCB as they might be entailed either in inflammation

(Hanisch et al., 2001) or phagocytosis (Sun et al., 2008; Tanaka et al., 2009). In fact, we

demonstrate that MAPKs phosphorylation is an essential step for NF-κB nuclear

translocation. Furthermore, our results reveal the induction of cyclooxygenase (COX)-2

expression and matrix metalloproteinase (MMP)-2 and -9 activity in a later phase of

microglial response to UCB, implicating these events in the overall deleterious and

inflammatory response. We provide evidence that UCB-induced cytokine secretion may

participate in MMP activation and hypothesize that these events might be reciprocally

regulated, further contributing to the complex network of microglia activation process.

Chapter II

52

Taken together, these results strongly imply a multiple response of microglia to UCB,

suggesting that those cells are relevant partners to consider during bilirubin

encephalopathy.

2. Material and Methods

2.1. Chemicals Dulbecco’s modified Eagle’s medium-Ham’s F12 medium (DMEM-Ham’s F-12),

Opti-MEM medium, fetal bovine serum (FBS), L-glutamine, sodium pyruvate and non-

essential aminoacids (NEA) were purchased from Biochrom AG (Berlin, Germany).

Antibiotic antimycotic solution (20X), human serum albumin (HSA; fraction V, fatty acid

free), bovine serum albumin (BSA), Hoechst 33258 dye, biotinylated tomato lectin

(Lycopersicon esculentum), avidin-fluorescein isothiocyanate (FITC), avidin-

tetramethylrhodamine isothiocyanate (TRITC), fluorescent latex beads 1μm (2.5%),

mouse anti-β-actin, FITC-labelled goat anti-rabbit IgG, rabbit anti-glial fibrillary acidic

protein (GFAP), TRITC-labelled goat anti-rabbit IgG, Coomassie Brilliant Blue R-250 and

propidium iodide (PI) were from Sigma Chemical Co. (St. Louis, MO). UCB was also

obtained from Sigma and purified according to the method of McDonagh and Assisi

(1972). Trypsin/Ethylenediamine tetraacetic acid (EDTA) solution (0.25% trypsin, 1 mM

EDTA in Hank’s balanced salt solution) and Alexa Fluor 594 chicken anti-goat IgG were

purchased from Invitrogen Corporation (Carlsbad, CA).

FuGENE HD Transfection Reagent was acquired from Roche Molecular

Biochemicals (Mannheim, Germany); Dual Luciferase reporter assay system was from

Promega (Madison, WI, USA); and caspase-3, -8 and -9 substrates, Ac-DEVD-pNA, Ac-

IETD-pNA and Ac-LEHD-pNA, respectively, were purchased from Calbiochem (San

Diego, CA). Concentrated solutions (10 mM) of MAPK pathways inhibitors SB203580

(p38 MAPK inhibitor; Calbiochem, La Jolla, CA, USA), and U0126 [Extracellular signal

regulated kinase (ERK1/2)-upstream inhibitor; Promega, Madison, WI, USA], were

prepared in dimethylsulfoxide.

Recombinant rat IL-1β and DuoSet® ELISA kits were from R&D Systems, Inc.

(Minneapolis, MN, USA). Nitrocellulose membrane, Hyperfilm ECL and Horseradish

peroxidase-labelled goat anti-mouse IgG were obtained from Amersham Biosciences

(Piscataway, NJ, USA). LumiGLO®, Cell lysis buffer®, rabbit anti-phosphorylated-p38

(P-p38) and rabbit anti-phosphorylated-ERK 1/2 (P-ERK1/2) were from Cell Signalling

(Beverly, MA, USA).

Mouse anti-phosphorylated-c-Jun N-terminal kinase (P-JNK1/2), rabbit anti-p65

NF-κB subunit and horseradish peroxidase-labelled goat anti-rabbit IgG were from Santa


53

Cruz Biotechnology (Santa Cruz, CA, USA). Goat anti-Ionized calcium-binding adaptor

molecule 1 (Iba1) was from Abcam (Cambridge, UK).

All other chemicals were of analytical grade and were purchased from Merck

(Darmstadt, Germany).

2.2. Primary culture of microglia Animal care followed the recommendations of European Convention for the

Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes

(Council Directive 86/609/EEC) and National Law 1005/92 (rules for protection of

experimental animals). All animal procedures were approved by the Institutional Animal

Care and Use Committee. Every effort was made to minimize the number of animals

used and their suffering.

Mixed glial cultures were prepared from 1-to-2 day-old Wistar rats as previously

described (McCarthy and de Vellis, 1980), with minor modifications (Gordo et al., 2006).

Cells (4 x 105 cells/cm2) were plated on uncoated 12 or 6-well tissue culture plates

(Corning Costar Corp., Cambridge, MA) in culture medium (DMEM-Ham’s F-12 medium

supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, nonessential amino acids

1X, 10% FBS, and 1% antibiotic-antimycotic solution) and maintained at 37ºC in a

humidified atmosphere of 5% CO2.

Microglia were isolated as previously described by Saura et al. (2003). Briefly,

after 21 days in culture, microglia were obtained by mild trypsinization with a trypsin-

EDTA solution diluted 1:3 in DMEM-Ham’s F12 for 20–45 min. The trypsinization

resulted in detachment of an upper layer of cells containing all the astrocytes, whereas

the microglia remained attached to the bottom of the well. The medium containing

detached cells was removed and replaced with initial mixed glial-conditioned medium.

Twenty-four hours after trypsinization, the attached cells were subjected to the different

treatments. The use of 21-days-in-vitro cells intents to achieve the maximal yield and

purity of the cultures. In fact, astrocyte contamination was less than 2%, as assessed by

immunocytochemical staining with a primary antibody against GFAP followed by a

species-specific fluorescent-labelled secondary antibody. Microglia were counterstained

with a biotinylated tomato lectin (Lycopersicon esculentum), using a 1:166 dilution in 1%

Triton X-100® in phosphate-buffered saline (PBS) overnight at 4ºC followed by 1 h

incubation at room temperature with avidin-TRITC in a 1:100 dilution in PBS and the

nuclei immunostained with Hoechst dye 33258. Thus, the high purity level of microglia

cultures excludes interference of contaminating astroglial cells.

Chapter II

54

2.3. Cell treatment Microglial cells were incubated in the absence (control) or in the presence of 50

µM UCB plus 100 µM HSA, from 5 min to 48 h, at 37ºC. A UCB stock solution (10 mM)

was prepared in 0.1 M NaOH immediately before use and the pH of the incubation

medium was restored to 7.4 by addition of equal amounts of 0.1 M HCl. All the

experiments with UCB were performed under light protection to avoid photodegradation.

To study the role of MAPK pathways in microglial response to UCB, cells were

pretreated for 20 min with 10 µM of the MAPK inhibitors prior to UCB stimulation:

SB203580, a selective inhibitor of p38 MAPK and U0126, a selective inhibitor of the

MAPK kinases (MEK)1/2, upstream kinases in the ERK1/2 pathway.

The involvement of IL-1β in MMP activation was investigated by treating microglia

with 2 ng/mL recombinant rat IL-1β or vehicle alone, in the presence of 100 µM HSA, for

30 min and 1 h at 37ºC. The selected concentration of cytokine was based on the

maximal levels obtained in our culture model upon UCB stimulation.

2.4. Measurement of cytokine release Aliquots of the cell culture media were collected at the end of the incubations and,

after removal of cellular debris by short centrifugation, placed in a 96-well microplate and

assessed in triplicate for TNF-α, IL-1β and IL-6 with specific DuoSet® ELISA

Development kits from R&D Systems, according to the manufacturer’s instructions.

Results were expressed as pg/mL.

2.5. Western blot assay Western blot assay was carried out as usual in our lab (Fernandes et al., 2006).

Briefly, total protein was extracted from primary microglia using Cell lysis buffer®. Protein

extracts were separated on a 10% sodium dodecyl sulphate-polyacrylamide gel

electrophoresis (SDS-PAGE). Following electrophoretic transfer onto a nitrocellulose

membrane and blocking with 5% milk solution, the blots were incubated with primary

antibody overnight at 4ºC [anti-P-p38 MAPK (1:1000), anti-P-ERK1/2 (1:1000), anti-P-

JNK1/2 (1:200), rabbit anti-COX-2 (1:1000) or anti-β-actin (1:10000) in 5% (w/v) bovine

serum albumin] and with horseradish peroxidase-labelled secondary antibody [anti-

mouse (1:5000) or anti-rabbit (1:5000)] for 1 h at room temperature. Protein bands were

detected by LumiGLO® and visualized by autoradiography with Hyperfilm ECL.


55

2.6. Detection of NF-κB activation

To assay the transcriptional activity of NF-κB, reporter gene analysis was applied.

A reporter plasmid under control of NF-κB binding sites was provided by Dr. Guy

Haegeman (Flanders Interuniversity Institute for Biotechnology and University of Gent,

Belgium). NF-κB-dependent reporter plasmids, p(IL6κB)350hu.IL6P-luc+, contain three

NF-κB binding sites in the promoter region, while NF-κB-independent plasmids,

p50hu.IL6P-luc+, do not (Vanden Berghe et al., 1998). These reporter genes were

introduced into microglial cells using FuGene HD Transfection Reagent. After 24 h of

transfection, cells were treated with 50 µM UCB plus 100 µM HSA from 30 min to 4 h, at

37ºC. Luciferase assays were carried out using a Dual Luciferase Reporter Assay

System (Promega), according to the instructions in manufacturer’s manual. Firefly and

renilla luciferase activities were measured using a luminometer (Berthold Technologies,

Wildbad, Germany). Firefly luciferase activity value was normalized to renilla luciferase

activity value from pSV-Sport-Rluc plasmid. Readings of promoter activities of NF-κB-

independent plasmids, p50hu.IL6P-luc+ and p1168hIL6m NF-κB-luc (plasmid presenting

a mutation in NF-κB binding sites), were also performed. Results were presented as fold

change of the relative luciferase activity compared to the respective control.

For immunofluorescence detection of NF-κB nuclear translocation, cells were

fixed for 20 min with freshly prepared 4% (w/v) paraformaldehyde in PBS and a standard

immunocytochemical technique was performed using a polyclonal rabbit anti-p65 NF-κB

subunit antibody (1:200) as the primary antibody and a FITC-labelled goat anti-rabbit

antibody (1:160) as the secondary antibody. To identify the total number of cells,

microglial nuclei were stained with Hoechst 33258 dye as previously described.

Fluorescence was visualized using a Leica DFC490 camera adapted to an AxioSkope®

microscope (Zeiss). Pairs of U.V. and green-fluorescence images of ten random

microscopic fields (original magnification: 400X) were acquired per sample. NF-κB-

positive nuclei (identified by localization of the NF-κB p65 subunit staining exclusively at

the nucleus) and total cells were counted (>500 cells per sample) to determine the

percentage of NF-κB-positive nuclei. Results were expressed as fold change vs.

respective control.

2.7. Morphological Analysis For morphological analysis, cells were fixed as described above and a standard

indirect immunocytochemical technique was carried out using a primary antibody raised

against Iba-1 (goat, 1:500) and a secondary Alexa Fluor 594 chicken anti-goat antibody

Chapter II

56

(1:200). Fluorescent images were acquired using a Leica DFC490 camera attached to

an AxioSkope® microscope (Zeiss).

2.8. Assessment of microglial phagocytic properties After treatment with UCB, cells were incubated with 0.0025% (w/w) 1 μm

fluorescent latex beads for 75 min at 37ºC and fixed with freshly prepared 4% (w/v)

paraformaldehyde in PBS. Labelling with tomato lectin was performed followed by avidin-

TRITC and the nuclei counterstained with Hoechst 33258 dye. U.V., green and red-

fluorescence images of fifteen random microscopic fields (original magnification: 630X)

were acquired per sample. The number of ingested beads per cell was counted in

approximately 250 cells per sample.

2.9. Gelatin zymography

Aliquots of culture supernatant were analyzed by SDS-PAGE zymography in

0.1% gelatine – 10% acrylamide gels under non-reducing conditions. After

electrophoresis, gels were washed for 1 h with 2.5% Triton X-100 (in 50 mM Tris pH7.4;

5 mM CaCl2; 1μM ZnCl2) to remove SDS and renature the MMP species in the gel. Then

the gels were incubated in the developing buffer (50 mM Tris pH7.4; 5 mM CaCl2; 1μM

ZnCl2) overnight to induce gelatine lysis. For enzyme activity analysis, the gels were

stained with 0.5% Coomassie Brilliant Blue R-250 and destained in 30% ethanol/10%

acetic acid/H2O. Gelatinase activity, detected as a white band on a blue background,

was quantified by computerized image analysis and normalized with total cellular protein.

2.10. Evaluation of microglial cell death Necrotic-like cell death was assessed by monitoring the cellular uptake of the

fluorescent dye propidium iodide [PI; 3,8-diamino-5-(3-(diethylmethylamino)propyl)-6-

phenyl phenanthridinium diiodide]. PI readily enters and stains non-viable cells, but

cannot cross the membrane of viable cells. This dye binds to double-stranded DNA and

emits red fluorescence (630 nm; absorbance 493 nm). Unpermeabilized adherent cells

cultured on coverslips were incubated with a 75 μM PI solution for 15 min in the absence

of light. Subsequently, cells were fixed with freshly prepared 4% (w/v) paraformaldehyde

in PBS and the nuclei immunostained with Hoechst 33258 dye.

Red-fluorescence and U.V. images of ten random microscopic fields (original

magnification: 400X) were acquired per sample and the percentage of PI positive cells

was counted and expressed as fold vs. respective control.


57

Activities of caspase-3, -8 and -9 were measured by a commercial colorimetric

method (Calbiochem, Darmstadt, Germany). Cells were harvested, washed with ice-cold

PBS and lysed for 30 min on ice in the lysis buffer [50 mM HEPES (pH 7.4); 100 mM

NaCl; 0.1% (w/v) CHAPS; 1 mM dithiothreitol (DTT); 0.1 mM EDTA]. The activities of

caspase-3, -8 and -9 were determined in cell lysates by enzymatic cleavage of

chromophore p-nitroanilide (pNA) from the substrate Ac-DEVD-pNA for caspase-3, Ac-

IETD-pNA for caspase-8 and Ac-LEHD-pNA for caspase-9, according to manufacturer’s

instructions. The proteolytic reaction was carried out in protease assay buffer [50 mM

HEPES (pH 7.4); 100 mM NaCl; 0.1% (w/v) CHAPS; 10 mM DTT; 0.1 mM EDTA; 10%

(v/v) glicerol], containing 2 mM specific substrate. Following incubation of the reaction

mixtures for 1 to 2 h at 37ºC, the formation of pNA was measured in a microplate reader

(PR 2100, BioRad Laboratories, Inc.) at λ= 405 nm with a reference filter at 620 nm.

Readings were normalized to total protein content determined using a protein assay kit

(Bio-Rad, Hercules, CA, USA) according to the manufacturer’s specification, and

expressed as fold change of respective control.

2.11. Statistical analysis Results of, at least, three different experiments were expressed as mean ±

S.E.M. Significant differences between two groups were determined by the two-tailed t-

test performed on the basis of equal and unequal variance as appropriate. Comparison

of more than two groups was done by ANOVA using Instat 3.05 (GraphPad Software,

San Diego, CA, USA) followed by multiple comparisons Bonferroni post-hoc correction.

Statistical significance was considered for a p value less than 0.05.

3. Results

3.1. UCB triggers IL-1β, TNF-α and IL-6 secretion following different temporal profiles

Supporting evidence reports reactive microglia as one of the main sources of pro-

inflammatory cytokines in the brain (Hanisch, 2002), which are known to exert

deleterious effects in nerve cells (Rothwell, 1999). Previous results suggested that UCB

is able to induce an inflammatory response by microglia (Gordo et al., 2006). Thus, we

intended to further characterize those inflammatory events by the evaluation of the

temporal secretion profile of IL-1β, TNF-α and IL-6. In Figure II.1 it can be observed that

UCB stimulates cytokine release in a time-dependent manner but following different

temporal profiles. In fact, TNF-α and IL-1β seem to be the first to be up-regulated, as an

increase in those cytokine levels in culture supernatants can be observed as early as 2 h

Chapter II

58

after the addition of 50 μM UCB. However, while peak values for TNF-α are reached at 4

h of UCB exposure (changing from 365 pg/mL for control conditions to 520 pg/mL for

UCB, p<0.05) and decline gradually along time of exposure (although an additional

increase is noticed at 24 h), the maximum release of IL-1β was only achieved at 12 h,

but in a much higher amount (ranging from 980 pg/mL for control conditions to 1700

pg/mL for UCB, p<0.05). On the other hand, IL-6 secretion was only noticed from 2 h on,

reaching peak levels at 8 h of UCB exposure (shifting from 1650 pg/mL for control

conditions to 2050 pg/mL for UCB, p<0.01) and decreasing thereafter. These results

seem to indicate that microglia responds to UCB stimulus with a rather inflammatory

profile which is manifested for prolonged incubation periods. As earlier results on

astrocytes demonstrated that UCB-induced cytokine secretion involves MAPK and NF-

κB activations (Fernandes et al., 2007; Fernandes et al., 2006) we sought to verify if the

same inflammatory signalling pathways are maintained by microglia.

Fig. II.1. UCB induces the release of TNF-α, IL-1β and IL-6 by microglia following different temporal profiles. Rat cortical microglial cells were treated with 50 µM UCB in the presence of 100 µM HSA for the indicated time periods. TNF-α, IL-1β and IL-6 concentrations in the media were determined by ELISA and expressed as mean ± SEM cytokine release from four independent experiments performed in triplicate, after deduction of cytokine values in control assays. *p<0.05 and **p<0.01 vs. respective control.

3.2. p38 and ERK1/2 phosphorylation is elicited by UCB in microglia at an early time point

MAPKs have been reported by several studies to be involved in the production of

inflammatory mediators by microglia (Bhat et al., 1998; Lee et al., 2000; Waetzig et al.,

2005), but their involvement on UCB microglial stimulation is still not known. So, we

assessed the phosphorylated (activated) forms of all three MAPKs (p38, ERK1/2 and

JNK1/2) in total cell lysates of UCB-exposed microglia, by western blot, using specific

antibodies.


59

As shown in Figure II.2, upon UCB stimulation, P-p38 and P-ERK1/2 expression

were significantly up-regulated in a rapid but transient manner. A 1.4-fold induction

(p<0.05 vs. control) was observed for P-p38 as early as 15 min after UCB exposure, and

this activation was sustained until 30 min of incubation (1.3-fold, p<0.05 vs. control),

fading afterwards. A second activation peak was observed after 2 h of exposure (1.4-

fold, p<0.05), declining to control levels at 6 h. In regard to P-ERK1/2, a 1.2-fold increase

(p<0.05 vs. control) was observed at 15 min of exposure and, as for P-p38, a second

peak was observed, but now smaller (1.12 -fold, p<0.05) and at 1 h of exposure,

declining from this point on. A weaker increase in P-JNK1/2 is perceived at 15 and 30

min of UCB exposure; however, this effect is not significantly different from the

respective controls.

Figure II.2. MAPKs activation is elicited by UCB in microglia. Rat cortical microglia were exposed to 50 µM UCB in the presence of 100 µM HSA for the indicated time periods. Total cell lysates were analysed by western blotting with antibodies specific for the phosphorylated forms of the three MAPKs, P-p38, P-ERK1/2 and P-JNK1/2. (A) Representative results of one experiment are shown. Similar results were obtained in three independent experiments. (B) The intensity of the bands was quantified by scanning densitometry, standardized with respect to β-actin protein expression and expressed as mean ± SEM fold change compared with control conditions. *p<0.05 vs. respective control.

Chapter II

60

Prolonging UCB incubation to longer periods did not modify the pattern of MAPK

activation (data not shown). These results indicate that MAPK activation is a rather early

event on microglial activation, seeming to involve two activation peaks. Next we found

interesting to check if this activation could be followed by the engagement of NF-κB

nuclear translocation.

3.3. NF-κB signalling pathway is triggered in UCB-activated microglia

NF-κB is described as a convergent point of signalling for microglial activation by

cytokines and other stressors (Glezer et al., 2007) and its implication in the inflammatory

response induced by UCB in astrocytes has already been established (Fernandes et al.,

2006). Hence, we examined the effect of UCB on NF-κB transactivation in microglial

cells by gene reporter assay (Figure II.3A). The results indicated that UCB markedly

induced NF-κB activation at 15 and 30 min of exposure (1.4-fold, p<0.01 for both time

points). It should be stated that readings of the promoter activities of p50hu.IL6P-luc+

and p1168hIL6m NF-κB-luc plasmids (empty and mutated vectors, respectively) showed

no change in the presence or absence of UCB (data not shown), thus validating the

assay.

To further confirm the activation of this signalling pathway we investigated NF-κB

activation in microglia exposed to UCB at various time points by the immunochemical

assessment of the cytoplasmic or nuclear localization of p65 NF-κB subunit. Interestingly

we found NF-κB translocation into the nucleus to be significantly increased from 15 min

to 2 h of exposure when compared to the respective controls (Figure II.3B-C), which is in

line with our previous observations regarding NF-κB transcriptional activation. Maximum

levels of nuclear NF-κB were observed at 30 min (2.2-fold, p<0.01), while from 4 h

onwards NF-κB was mostly localized in the cellular cytoplasm. These results follow the

ones from MAPK activation, suggesting that, as previously verified in astrocytes, both

events are connected.


61

Fig. II.3. UCB activates NF-κB signalling pathway in microglia. Rat cortical microglia were exposed to 50 µM UCB in the presence of 100 µM HSA for the indicated time periods. (A) Microglial cells were transiently transfected with NF-κB-dependent luciferase plasmids and control plasmids. Relative luciferase activities were plotted as fold change of respective controls. To further confirm NF-κB activation, cells were fixed and immunostained with an antibody against p65 NF-κB subunit. (B) Representative results of one experiment are shown. Scale bar, 20 μm. (C) The percentage of NF-κB-positive nuclei was calculated and expressed as fold change vs. respective control. Results are mean ± SEM from three independent experiments performed in triplicate. *p<0.05 and **p<0.01 vs. respective control.

3.4. UCB-induced NF-κB translocation depends on both ERK1/2 and p38

To assess whether MAPKs phosphorylation is an essential step for UCB-evoked

NF-κB translocation, we investigated this event after exposure of microglia to UCB alone

or in combination with the MAPK inhibitors SB203580 (p38 selective inhibitor) and

UO126 [(MEK)1/2 selective inhibitor, upstream kinases in the ERK1/2 pathway]. The use

of 30 min and 1 h incubation periods was based on previous results showing that

maximal translocation of NF-κB to the nucleus in UCB-challenged microglia occurs

between 30 min and 4 h.

Chapter II

62

As expected, pre-incubation with SB203580 and U0126 completely abrogated

UCB-stimulated NF-κB nuclear translocation after 30 min (p<0.01) and 1 h (p<0.05) of

UCB stimulation (Figure II.4), thus providing proof of concept that p38 and ERK1/2

phosphorylation are required for the engagement of NF-κB pathway upon UCB

exposure.

Fig. II.4. Phosphorylation of p38 and ERK1/2 is essential for UCB-evoked NF-κB activation. Rat cortical microglia were exposed for 30 min and 1 h to 50 µM UCB alone or in combination with 10 µM of the p38 inhibitor SB203580 or the ERK1/2 inhibitor U0126. Cells were fixed and immunostained with an antibody against p65 NF-κB subunit. (A) Representative results of one experiment are shown. Scale bar, 20 μm. (B) The percentage of NF-κB-positive nuclei was calculated and expressed as fold change vs. respective control. Results are mean ± SEM from three independent experiments performed in triplicate. **p<0.01 vs. respective control, §p<0.05 and §§p<0.01 vs. UCB alone.


63

So far our results have described the engagement of early inflammatory

signalling pathways that culminate in the acquisition of an inflammatory phenotype

characterized by the release of proinflammatory mediators. We next intended to

characterize UCB-stimulated microglia at a morphological level, in order to verify the

acquisition of this activated state.

3.5. Microglia depict morphological changes upon UCB stimulation Modification of microglial morphology is one of the hallmarks of its activation

profile and has been widely used to categorize different activation states (Chew et al.,

2006; Kim and de Vellis, 2005; Lynch, 2009; Raivich et al., 1999). For that reason, we

evaluated the morphology and reactivity of UCB-stimulated microglia by

immunocytochemistry after 4 and 24 h incubation periods. Our results indicate that, after

a short UCB exposure, microglia shifted from an elongated morphology to a large and

amoeboid shape (Figure II.5). This phenotype is characteristic of activated or reactive

microglia (Nakajima and Kohsaka, 2004). In contrast, for longer exposure periods,

microglia display fragmented and condensed cytoplasm, a feature described by other

authors (Fendrick et al., 2007; Hasegawa-Ishii et al., 2010) as cytorrhexis, indicative of

microglia degeneration and senescence.

Fig. II.5. UCB-stimulated microglia show reactive morphological changes. Rat cortical microglia were exposed to 50 µM UCB in the presence of 100 µM HSA for the indicated time periods. Cells were fixed and immunostained with an antibody against Iba1 reactivity marker. Representative results of one experiment are shown. Scale bar, 20 μm.

Interestingly, the inflammatory phenotype previously described occurs after

prolonged UCB incubation periods. However, activation features were also observed for

shorter stimulations. For that matter, we sought to evaluate the phagocytic properties of

UCB-challenged microglia and, more importantly, to verify whether this reactive

phenotype occurred prior, simultaneously or after the inflammatory response triggered by

UCB, in order to further characterize the chronologic events of UCB microglial activation.

Chapter II

64

3.6. UCB differently modulates microglial phagocytosis depending on exposure time

Phagocytosis is one of the main features of microglial activation dumping cell

debris prior to cell regeneration, and can be involved in the pathogenesis of several brain

dysfunctions (Neumann et al., 2009). However, this microglial property was never

investigated under UCB stimulation. As can be seen in Figure II.6, a sharp increase in

the uptake of fluorescent latex beads by UCB-stimulated microglia occurs from 2 h on,

reaching a maximum peak after 4 h of exposure, (~50%, p<0.05). This is a strong

evidence that UCB is able to induce microglial phagocytic properties in a rather early

time of exposure.

Fig. II.6. Microglial phagocytosis is differently modulated by UCB. Rat cortical microglia were exposed to 50 µM UCB in the presence of 100 µM HSA for the indicated time periods and incubated with 1 μm fluorescent latex beads as described in Methods. (A) Representative results of one experiment are shown. Scale bar, 20 μm. (B) Results are expressed as number of ingested beads per cell (± SEM) from three independent experiments. *p<0.05 vs. respective control.


65

Unspecific entry of latex beads due to UCB-induced cell permeabilization was

excluded by performing the phagocytosis assay in microglial cells incubated with UCB for

4 h and simultaneously determining PI uptake. Dead cells did not appear to engulf

particles (data not shown). Interestingly, prolonging UCB incubation for 8 and 12 h

slightly reverts this phagocytic ability although it approached control values for 24 h of

exposure. This may indicate that, after an initial phagocytic reaction, microglial cells

shifted to the previously observed inflammatory response to UCB stimulus. To add on

the characterization of microglial behaviour we advanced to the evaluation of other

markers of its inflammatory response such as MMPs activity and COX-2 expression.

3.7. Release of active MMPs is enhanced upon UCB stimulation of microglia

MMPs are a family of proteases with many important roles in normal development

although they may also participate in several neuronal diseases such as multiple

sclerosis, ischemia and neuroinflammation given their ability to degrade the basal lamina

surrounding the blood-brain barrier allowing infiltration of immune cells and, thus,

aggravating inflammatory reactions in the CNS (Michaluk and Kaczmarek, 2007;

Rosenberg, 2002). Since microglia have been reported to secrete active MMPs further

contributing to neuronal injury (Kauppinen and Swanson, 2005; Woo et al., 2008), and

given the fact that cytokines are reported to stimulate MMPs secretion and activity

(Gottschall and Yu, 1995; Lin et al., 2009; Vincenti and Brinckerhoff, 2007), we intended

to evaluate the levels of active MMPs secreted by these cells in response to UCB and to

verify if this activation could be ascribed to UCB-induced IL-1β.

Cell supernatants collected after UCB incubation were subjected to gelatin

zymography for the assessment of MMP-2 and MMP-9 activity levels. As can be seen in

Figure II.7 there is a slight but significant increase in the activity of MMP-2 and MMP-9

(1.07-fold and 1.08-fold, p<0.05, respectively) after a prolonged exposure time (24 h) to

UCB.

Chapter II

66

Fig. II.7. MMP-2 and MMP-9 activities are enhanced upon UCB stimulation. Culture supernatants from rat cortical microglial cells were harvested after incubation with 50 µM UCB in the presence of 100 µM HSA or with 2 ng/mL IL-1β for the indicated time periods and subjected to zymography as described in Methods. (A) Representative gels of one experiment are reported. MMP-2 and MMP-9 were identified by their apparent molecular mass of 67 and 92 kDa, respectively. (B) The intensity of the bands was quantified by scanning densitometry, standardized with respect to total protein content and expressed as mean ± SEM fold change compared with control conditions. *p<0.05 and **p<0.01 vs. respective control.

Since this event occurs after the onset of cytokine secretion elicited by microglia,

a possible regulation of this event by inflammatory mediators could be occurring. In fact,

stimulation of microglia with 2 ng/mL of IL-1β (which correspond to maximal levels

obtained in our culture model upon UCB stimulation) increases significantly MMP-2 and

MMP-9 activity at both 12 (1.3-fold and 1.2-fold, p<0.05 and p<0.01, respectively) and 24

h of exposure (1.2-fold and 1.3-fold, p<0.01, respectively).

3.8. UCB-stimulated microglia evidence enhanced COX-2 expression COX-2 is the enzyme responsible for the production of prostanoids such as

prostaglandin E2 (PGE2), which is known to be involved in the initiation and propagation

of the immune response (de Oliveira et al., 2008). The expression of this enzyme can be

induced by lipopolysaccharide (LPS) and cytokines (Levi et al., 1998) and has been

proven to be elevated in Alzheimer’s disease (Yokota et al., 2003) and ischemia

(Iadecola et al., 1999). As depicted in Figure II.8, a significant up-regulation of COX-2

expression was only noticed after 12 and 24 h of UCB exposure (1.13-fold, p<0.01 and


67

1.18-fold, p<0.05, respectively), when compared to the respective controls, again

suggesting a later response of microglia to UCB, in line with the previous results.

Finally, we were interested in examining how UCB interaction affected microglial

cell survival.

Fig. II.8. COX-2 expression is up-regulated by UCB in microglia. Rat cortical microglia were exposed to 50 µM UCB in the presence of 100 µM HSA for the indicated time periods. Total cell lysates were analysed by western blotting. (A) Representative results of one experiment are shown. Similar results were obtained in three independent experiments. (B) The intensity of the bands was quantified by scanning densitometry, standardized with respect to β-actin protein and expressed as mean ± SEM fold change compared with control conditions. *p<0.05 vs. respective control.

3.9. UCB reduces microglial viability leading to loss of membrane integrity and increased caspase activity

To evaluate the necrotic-like cell death we used the uptake of the fluorescent dye

PI as an indicator of membrane integrity and cell damage since this polar substance can

only enter dead or dying cells. To address the possible involvement of the apoptotic

pathways in microglial demise we determined the relative levels of caspases activity,

since these proteases have been traditionally viewed as central regulators of apoptosis

(Fink and Cookson, 2005). As depicted in Figure II.9, UCB stimulation only arouses

increased PI uptake from 4 to 12 h of exposure, reaching maximum significance at 8 h.

Accordingly, the activities of initiator caspases -8 and -9 were found to be significantly

elevated in response to UCB from 2 to 12 h of exposure reaching maximum activity at 6

h (Figure II.10) while effector caspase-3 was significantly increased at a relatively later

time point (from 4 to 12 h of exposure). It is interesting to notice that cell death seems to

occur on the onset of inflammatory response and when phagocytic activity declines,

again suggesting a possible double response from microglia to UCB.

Chapter II

68

Fig. II.9. UCB induces microglial decreased viability and membrane disruption. Rat cortical microglia were exposed to 50 µM UCB in the presence of 100 µM HSA for the indicated time periods and incubated with 75 μM PI as described in Methods. The percentage of PI-positive cells was calculated and expressed as fold vs. respective control. Results are mean ± SEM from three independent experiments performed in triplicate *p<0.05 and **p<0.01 vs. respective control.

Fig. II.10. Microglial apoptotic cell death is elicited by UCB. Rat cortical microglia were exposed to 50 µM UCB in the presence of 100 µM HSA for the indicated time periods. The activities of caspases -3, -8 and -9 were determined in cell lysates by enzymatic cleavage of chromophore p-nitroanilide (pNA) from specific substrates. Results are expressed as fold of respective control at each time point. Data are means ± SEM from at least three independent experiments. *p<0.05 and **p<0.01 vs. respective control.


69

4. Discussion In this paper we describe, for the first time, different activation stages of microglia

in the presence of UCB, since these cells display both phagocytic and inflammatory

phenotypes. Indeed, this study is original in depicting the increased phagocytic

properties of microglia upon UCB stimulation. In addition, our group was also the first to

implicate microglial cells in the inflammatory response elicited by UCB (Gordo et al.,

2006). Thus, in this study we further investigated the activation profile of microglia under

UCB stimulation, by the evaluation of some of its characteristical features, and the

signalling events involved in cell response. In fact, previous studies performed by our

group have proven that UCB induces immunological responses in astrocytes by the

activation of inflammatory pathways and secretion of glutamate (Fernandes et al., 2006;

Fernandes et al., 2004), and also that UCB is neurotoxic (Falcão et al., 2007; Silva et al.,

2001). Accordingly, it is conceivable that increased microglia reactivity may further

contribute to neuronal injury during hyperbilirubinemia.

Microglia contribute to both innate and adaptive immune responses in the brain

(Chew et al., 2006). As innate immune cells, they constitute the first line of defence

against invading microorganisms. The hallmark indicators of such response are the

production of pro-inflammatory cytokines, the up-regulation of cell surface antigens and

phagocytosis (Town et al., 2005). In addition, phagocytosis of debris by microglia can be

beneficial in several pathological conditions such as multiple sclerosis (Takahashi et al.,

2007) and Alzheimer’s disease (Simard et al., 2006) as it restricts lesion extension and

facilitates tissue recovery.

The fact that UCB may alter the function of various cells of the immune system

(both in vivo and in vitro) seems to be firmly established and a wide range of

immunosuppressive effects on peripheral immune cells are summarized by Vetvicka et

al. (1991) such as alterations on antigen expression, chemotaxis, bactericidal activity,

proliferative response of T lymphocytes, or antibody production. On the other hand, an

increase in phagocytosis of both peripheral blood granulocytes and monocytes after

UCB treatment was reported by Miler et al. (1985). Thus, a rather contradictory

immunosuppressive – immunostimulant status seems to be observed upon UCB

challenge that might be explained by dose or time-dependent effect (Vetvicka et al.,

1991).

Our findings demonstrate that, in conditions that intend to mimic a mild

hyperbilirubinemia, enhancing of microglial phagocytic properties by UCB is an early, but

transient, event that seems to be lost with increased time of exposure. Thus, we may

assume that phagocytosis is the first response towards UCB insult and may constitute a

neuroprotective measure.

Chapter II

70

Various conditions have been shown to greatly modify microglial phagocytic

activity, such as cytokines (Koenigsknecht-Talboo and Landreth, 2005), and LPS (Sun et

al., 2008), among others. Interestingly, the study performed by von Zahn et al. (1997)

reports an induction of nearly two-fold increase in the uptake of uncoated latex particles

by TNF-α-stimulated microglia, substantiating this cytokine as an autocrine activator of

microglial immune functions. Indeed, UCB-activated microglia are reportedly one of the

main sources of TNF-α, even when compared to astrocytes (Brites et al., 2009). Similarly

to what we already observed for astrocytes (Fernandes et al., 2006), our results point to

TNF-α as the first cytokine to be released by microglia upon UCB challenge, and,

remarkably, its temporal profile of secretion is rather paralleled by the observed

phagocytic alterations. TNF-α secretion reaches a maximum level at 4 h of UCB

exposure, when microglial phagocytic properties are significantly increased and a decline

in UCB-induced TNF-α release is observed from this point on, coinciding with decreased

phagocytosis elicited by UCB.

Besides its role as microglial phagocytosis inducer, several experiments have

also implicated TNF-α in demyelination (Akassoglou et al., 1998) and neuronal

degeneration (Allan and Rothwell, 2003; Silva et al., 2006), and this cytokine, along with

IL-1β, participates in astrogliosis (Hanisch, 2002). IL-1β is involved in fever induction and

edema, stimulation of COX-2, release of nitric oxide (NO) and free radicals (Rothwell,

1999), also participating in the recruitment of circulating leukocytes into the CNS due to

its ability to up-regulate the expression of adhesion molecules and chemokine synthesis

(Lee and Benveniste, 1999; Sedgwick et al., 2000). IL-6 can have both pro and anti-

inflammatory functions and is produced in the early phases of CNS insult (Raivich et al.,

1999). Our results clearly imply microglia as an important player in the inflammatory

response instigated by UCB, since the observed early release of TNF-α, previously

discussed, is followed by a later but intense secretion of IL-6 and an even stronger

induction of IL-1β.

Strikingly, UCB seems to induce a major release of pro-inflammatory cytokines in

a time period in which phagocytosis is already absent. Recent reports have, in fact,

substantiated the existence of a non-phlogistic (non-inflammatory) phagocytic response

from microglia (Neumann et al., 2009), triggered by apoptotic stimuli and potentially

mediated by phosphatidylserine receptors (PRs) and triggering receptor expressed on

myeloid cells-2 (TREM2) (Hsieh et al., 2009; Takahashi et al., 2005). Additionally, IL-1β

and PGE2 were shown to suppress microglial ability to phagocytise insoluble fibrillar β-

amyloid deposits, suggesting that a pro-inflammatory milieu inhibits this type of

phagocytosis (Koenigsknecht-Talboo and Landreth, 2005).


71

Our findings also suggest a role for MMPs in UCB-induced microglia reactivity,

since their activity is enhanced upon prolonged exposure periods to this molecule. MMP-

9 has been associated with glutamate dysfunction (Michaluk and Kaczmarek, 2007) and

its release can be a cause of microglia-induced neuron death (Kauppinen and Swanson,

2005). In addition, inhibition of gelatinases (MMP-2 and -9) showed efficacy in reducing

neural injury and dampening neuroinflammation (Leonardo et al., 2008). Thus, these

proteases seem to actively participate in inflammatory events and their activity is very

tightly regulated (Sternlicht and Werb, 2001). Cytokines are firmly established inducers

of MMP expression and secretion (Gottschall and Yu, 1995; Ito et al., 1996), and the

induction of MMPs has been shown to be mediated by MAPKs, NF-κB and activator

protein-1 signalling pathways (Lin et al., 2009; Shakibaei et al., 2007; Vincenti and

Brinckerhoff, 2007; Woo et al., 2008). Interestingly, in our study model, MMPs enhanced

activity occurs at a later time of exposure, when MAPKs and NF-κB activation as well as

cytokine secretion have already taken place, suggesting that these events might be

involved in the activation of MMPs induced by UCB. Moreover, active MMPs may also

participate in the regulation of cytokine activity by promoting the secretion and activation

of these molecules (Chauvet et al., 2001; Kim et al., 2005; Nuttall et al., 2007; Woo et al.,

2008) or, on the other hand, by negatively regulating their biological activities (Ito et al.,

1996). As stated above, MMPs increased activity in UCB-stimulated microglia takes

place after the peak of cytokine secretion, suggesting a possible reciprocal regulation

between pro-inflammatory cytokines and MMPs, since the latter molecules could be

involved in the termination of the inflammatory response by means of the degradation of

IL-1β. Our results further address this issue since IL-1β demonstrated to intensely

elevate MMP-2 and -9 activation providing proof of concept that IL-1β secretion

produced upon UCB stimulation, is at least in part, responsible for MMP activation.

Discrepancy between the activation levels observed for UCB or IL-1β alone may be a

result of UCB-multiple cytokine activation which results in a pleiotropic regulatory loop,

absent in the second condition.

COX-2 expression can also be induced in microglia by several inflammatory

conditions. As can be seen in our results, UCB is able to induce up-regulation of COX-2

in microglial cells in a profile very similar to the observed for IL-6 and IL-1β, thus

contributing to the overall inflammatory environment described so far and again pointing

to an inflammatory response secondary to phagocytosis. Therefore, our studies suggest

a dual role for microglia upon UCB stimulation, shifting from a phagocytic and possibly

neuroprotective phenotype towards an inflammatory and deleterious one. This is

Chapter II

72

consistent with our findings demonstrating that microglia portray altered morphological

features after a prolonged UCB exposure, typical of an activated state.

As previously observed for astrocytes (Fernandes et al., 2006; Fernandes et al.,

2004) we show here that UCB stimulation of microglial cells also involves the activation

of MAPKs and NF-κB. MAPKs can be activated by a variety of different stimuli (Roux

and Blenis, 2004), and the engagement of this signalling pathway can lead to the

phosphorylation of several substrates, including transcription factors such as NF-κB,

which may ultimately lead to the enhanced transcription of genes encoding for pro-

inflammatory cytokines (Koj, 1996). Activation of p38 and ERK1/2 are regarded as

essential steps for cytokine induction since their involvement in TNF-α, IL-1β, IL-6, COX-

2 and inductible nitric oxide synthase (iNOS) expression in microglia has been widely

established (Bhat et al., 1998; Hanisch et al., 2001; Lee et al., 2000). Intriguingly,

MAPKs activation, particularly p38, seems to be also involved in the induction of

microglia phagocytosis (Sun et al., 2008; Tanaka et al., 2009).

In this regard our data indicate a rapid activation of p38 and ERK1/2 by UCB in

microglial cells, which occurs prior to the production of inflammatory mediators

previously reported. In fact, MAPKs activation by UCB in microglia is triggered at a much

earlier stage than in astrocytes (Fernandes et al., 2006), reinforcing the greater

responsiveness of these glial cells during hyperbilirubinemia but also suggesting that the

early phagocytic response of microglia cells to UCB may be under the control of p38 and

ERK1/2 activation. In this case, the latter activation peak observed would engage the

pro-inflammatory cascade that results in IL-1β and IL-6 enhanced secretion, as well as

the induction of COX-2 and MMPs, a feature already observed for other immune cells

(Gong et al., 2008; Hwang et al., 1997).

Moving downstream on the intracellular signalling pathways is the original

observation that, as in astrocytes (Fernandes et al. 2007; Fernandes et al. 2006), NF-κB

activation is also present in microglia exposed to UCB. Interestingly, maximum activation

of NF-κB takes place during and after the early MAPKs phosphorylation and again prior

to the production of IL-1β, TNF-α and IL-6, postulating a possible involvement of NF-κB

in both phagocytic and inflammatory responses elicited by UCB in microglia. The

observations that NF-κB nuclear translocation in UCB-stimulated microglia is completely

abrogated when microglia are pretreated with p38 and ERK1/2 inhibitors provided an

unequivocal proof of MAPKs involvement in NF-κB engagement in UCB-challenged

microglia which had already been previously established by other authors in different

disease models (Wilms et al., 2003).


73

It isworthwhile to mention that cell viability and membrane integrity are

compromised from 4 h onwards, indicating increased cell damage induced by UCB on

microglial cells from this point onwards. In fact, UCB-induced apoptotic and necrotic

microglial cell death have already been established (Gordo et al., 2006). Our results

indicate that both the extrinsic and intrinsic apoptotic pathways are triggered culminating

in the activation of effector caspase-3 and consequently causing cell death. However,

cell death phenomena reach maximum peaks between 6 to 8 h but decreased for longer

incubation periods. Together with the findings described above these data portray an

interesting hypothesis for microglia response to UCB stimulus. So, it is conceivable that,

either a shift on global microglia reaction occurs, or there are two distinct sub-

populations of microglial cells displaying complementary activation features, one directed

at eliminating the damaged cells by phagocytosis, that died after engulfment of beads,

and another engaging a more delayed inflammatory response. In fact, fragmentation of

cytoplasm (cytorrhexis) which is suggested in our 24 h morphological observations, has

been pointed to be indicative of widespread microglial degeneration in amyotrophic

lateral sclerosis models (Fendrick et al., 2007). Degenerative changes in microglia such

as beading and clusters of fragmented twigs have also been demonstrated in the aged

brain (Hasegawa-Ishii et al., 2010). Which of the above mentioned hypotheses is the

more valid demands further elucidation and will clarify the multifaceted profile of

microglia activation under UCB stimulation. The complex network of UCB-induced

events in microglia, as well as the proposed interactions between them, is depicted in

Figure II.11.

In conclusion, our experiments evidence that phagocytosis is differently

modulated by UCB depending on the time of exposure, prevailing at an early time point,

which is followed by the release of inflammatory cytokines, and activation of MMP-2 and

-9, as well as of COX-2 activation. Thus, microglial phagocytosis and inflammatory

response stand out as important events prompted by UCB. To what extent the activation

of microglia by UCB has a beneficial or detrimental outcome is yet to be determined in

future studies, where the influence of other nerve cells will be evaluated. Nevertheless,

modulation of microglial activation seems to be a promising target in neonatal bilirubin

encephalopathy.

Chapter II

74

Fig. II.11. Schematic representation of time-dependent microglial activation induced by UCB. Upon UCB stimulation of microglial cells, MAPK and NK-κB signalling pathways are engaged, culminating in the generation of a phagocytic response followed by an inflammatory profile. Both phenotypes might alternate due to a reciprocal regulatory effect or to the existence of two different sub-populations engaging both types of response, being the phagocytic sub-population firstly extinguished and replaced by a rather inflammatory sub-population. This inflammatory profile is characterized by the increased release of pro-inflammatory cytokines TNF-α, IL-1β and IL-6, the up-regulation of COX-2 and enhanced activities of MMP-2 and MMP-9. Regulatory interactions between the UCB-induced events are portrayed in the figure.

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Chapter III

DYNAMICS OF NEURON-GLIA INTERPLAY UPON EXPOSURE TO UNCONJUGATED BILIRUBIN

Sandra L. Silva, Catarina Osório, Ana R. Vaz, Andreia Barateiro, Ana S. Falcão, Rui F. M. Silva, Dora Brites

Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL),

Faculdade de Farmácia, University of Lisbon,

Av. Professor Gama Pinto, Lisbon 1649-003, Portugal.

Journal of Neurochemistry, 2010 (submitted)

80

Acknowledgments This work was supported by PTDC/SAU-NEU/64385/2006 grant, from Fundação para a

Ciência e a Tecnologia (FCT), Lisbon, Portugal (to D.B.). S.L.S. was recipient of a PhD

fellowship (SFRH/BD/30326/2006) from FCT.

Glial and neuronal pathological alterations by unconjugated bilirubin

81

Abstract Microglia are the main players of the brain immune response acting as active

sensors that rapidly respond to injurious insults by shifting into different activated states.

Elevated levels of unconjugated bilirubin (UCB) induce cell death, immunostimulation

and oxidative stress in both neurons and astrocytes. We recently reported that microglia

phagocytosis phenotype precedes the release of pro-inflammatory cytokines upon UCB

exposure. We investigated whether and how microglia microenvironment influences the

response to UCB. Our findings, revealed that conditioned media derived from UCB-

treated astrocytes reduce microglial inflammatory reaction and cell death, suggesting an

attempt to refrain microglial overactivation. Conditioned medium from UCB-challenged

neurons, although down-regulating TNF-α and IL-1β promoted the release of IL-6 and

nitric oxide, the activation of matrix metalloproteinase-9, and cell death, as compared to

UCB-direct effects on microglia. Moreover, soluble factors released by UCB-treated

neurons showed to intensify the phagocytic properties manifested by microglia under

direct exposure to UCB. Results from neuron-microglia mixed cultures incubated with

UCB evidenced that sensitized microglia was able to prevent neurite outgrowth

impairment and cell death. In conclusion, our data indicate that stressed neurons signal

microglial clearance functions, but also overstimulate its inflammatory potential ultimately

leading to microglia demise. Keywords: Astrocyte- and neuron-conditioned media; Inflammatory reaction; Nerve cell

death; Neurite outgrowth and branching; Neuron-microglia mixed cultures; Nitrosative

stress; Phagocytosis; Unconjugated bilirubin.

Chapter III

82

1. Introduction

Hyperbilirubinemia is a common disorder of the brain that occurs in the neonatal

period (Dennery et al., 2001; Porter and Dennis, 2002) and has been associated with

minor neurologic dysfunction (Soorani-Lunsing et al., 2001), motor and auditory

impairment (Oh et al., 2003; Shapiro and Nakamura, 2001). In fact, recent findings have

strongly correlated auditory neuropathy disorder and acute bilirubin-induced neurotoxicity

(Saluja et al., 2010). Moreover, neonatal hyperbilirubinemia might be a vulnerability

factor for mental disorders (Dalman and Cullberg, 1999) such as schizophrenia

(Hayashida et al., 2009; Miyaoka et al., 2000). Interestingly, elevated levels of

unconjugated bilirubin (UCB) have also been associated with sepsis (Zhai et al., 2009)

and fever (Kaplan and Hammerman, 2005). Indeed, previous reports from our work

group have already demonstrated the immunostimulant properties of UCB in neurons

(Falcão et al., 2006) and, in a more marked fashion, in astrocytes (Falcão et al., 2005;

Fernandes et al., 2006; Fernandes et al., 2004) and microglia (Gordo et al., 2006; Silva

et al., 2010), thus reinforcing the immunostimulant effects of UCB.

Microglial cells have been acknowledged as the brain’s immune system

constituting a link between the central nervous system (CNS) – a barrier protected organ

– and the general immune system (Aloisi, 2001; Streit, 2002). Microglia can perform a

plethora of diverse functions that range from immune surveillance (Nimmerjahn et al.,

2005) and phagocytic debris clearance (in both physiologic and pathologic conditions)

(Cuadros and Navascues, 1998; Neumann et al., 2009; Streit, 2001), to the production of

inflammatory mediators and reactive oxygen and nitrosative species (ROS and RNS,

respectively) (Aloisi, 2001; Kim and de Vellis, 2005) which may contribute to nerve cell

dysfunction and death (Gibbons and Dragunow, 2006). Our group has recently shown

that microglia reacts to UCB by the acquisition of a phagocytic phenotype that shifts into

a rather inflammatory profile (Silva et al., 2010). Accordingly, some authors have actually

described microglia as active sensors that rapidly respond to microenvironmental

changes with the engagement of different reactivity profiles (Hanisch and Kettenmann,

2007).

Indeed, cellular interactions may be relevant for microglial reactivity. Neurons

have proven its role as key immune modulators in the brain as they can influence the

release of inflammatory or neurotrophic mediators form microglia (Biber et al., 2007; Lai

and Todd, 2008; Polazzi and Contestabile, 2006). Furthermore, damaged neurons can

signal microglia to engage phagocytic clearance, thus limiting injury extension (Petersen

and Dailey, 2004; Witting et al., 2000). Astrocytes can also attenuate microglial toxicity

(Giulian et al., 1993; Rozenfeld et al., 2003) and down-regulate its inflammatory reaction

(Eskes et al., 2003; Min et al., 2006) or, in opposite, further activate microglia through


83

increased ROS production (Yang et al., 1998). Finally, microglial reactivity is widely

recognized as a cause of cell demise (Gibbons and Dragunow, 2006; Munch et al., 2003;

Wang et al., 2005), that in some circumstances may also lead to a neuroprotective

outcome (Minghetti et al., 2005; Zhang and Fedoroff, 1996). Considering that interplay

between CNS cells may have such a strong and diverse influence on the cascade of

microglial responses, this study was undertaken to ascertain if UCB-activated astrocytes

or neurons could modulate the reactive response of microglia. Our findings indicate that

soluble factors released by UCB-treated astrocytes dampen microglial inflammatory

reaction upon UCB exposure. On the other hand, conditioned medium from neurons

incubated with UCB, although exerting a similar down-regulation in microglial production

of inflammatory cytokines, up-regulated matrix metalloproteinases (MMP) and nitric

oxide (NO) secretion. Furthermore, neurons were able to augment microglial phagocytic

abilities when facing UCB and ultimately increased UCB-induced microglial cell death.

Microglia-neuron interplay in mixed cultures upon UCB stimulation revealed a

neuroprotective role of microglia in preventing neurite impairment and necrotic-like cell

death by UCB. We hypothesize that UCB-injured neurons might be signaling microglia to

engage a phagocytic phenotype while hampering inflammatory reaction in an attempt to

constrain lesion extent.

2. Material and Methods

2.1. Chemicals Neurobasal medium, B-27 Supplement (50x), Hanks’ balanced salt solution

(HBSS), HBSS without Ca2+ and Mg2+, gentamicin (50 mg/mL), trypsin (0.5 g/L), Alexa

Fluor 594 chicken anti-goat IgG and Trypsin/Ethylenediamine tetraacetic acid (EDTA)

solution (0.25% trypsin, 1 mM EDTA in HBSS) were acquired from Invitrogen (Carlsbad,

CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), DMEM-Ham’s F12 medium,

fetal bovine serum (FBS), sodium pyruvate, non-essential aminoacids (NEA) and L-

glutamine, were purchased from Biochrom AG (Berlin, Germany). Antibiotic antimycotic

solution (20X), human serum albumin (HSA; Fraction V, fatty acid free), Hoechst 33258

dye, biotinylated tomato lectin (Lycopersicon esculentum), avidin-fluorescein

isothiocyanate (FITC), avidin-tetramethylrhodamine isothiocyanate (TRITC), fluorescent

latex beads 1 μm (2.5%), propidium iodide (PI), N-1-naphthylethylenediamine, and

Coomassie Brilliant Blue R-250 were purchased from Sigma Chemical Co. (St. Louis,

MO, USA). UCB was also obtained from Sigma and purified according to the method of

McDonagh and Assisi, (1972).

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DuoSet® ELISA kits were from R&D Systems, Inc. (Minneapolis, MN, USA). Goat

anti-ionized calcium-binding adaptor molecule 1 (Iba1) was from Abcam (Cambridge,

UK) and mouse anti-microtubule associated protein (MAP)-2 antibody was from

Chemicon (Temecula, CA, USA). FITC-labeled horse antibody anti-mouse was acquired

from Vector (Burlingame, CA, USA). Lactate dehydrogenase (LDH) cytotoxicity detection

kit was purchased from Roche Molecular Biochemicals (Manheim, Germany).



2.2. Primary culture of microglia Animal care followed the recommendations of European Convention for the



experimental animals). All animal procedures were approved by the Institutional Animal

Care and Use Committee. Every effort was made to minimize the number of animals

used and their suffering.

Mixed glial cultures were prepared from 1 to 2 day-old Wistar rats as previously

described (McCarthy and de Vellis, 1980), with minor modifications (Gordo et al., 2006).

Cells (4 X 105 cells/cm2) were plated on uncoated 12 or 6-well tissue culture plates

(Corning Costar Corp., Cambridge, MA, USA) in culture medium (DMEM-Ham’s F-12

medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, NEA 1X, 10%

FBS, and 1% antibiotic-antimycotic solution) and maintained at 37ºC in a humidified

atmosphere of 5% CO2.

Microglia were isolated as previously described by Saura et al., (2003). Briefly,

after 21 days in vitro (DIV), microglia were obtained by mild trypsinization with a trypsin-

EDTA solution diluted 1:3 in DMEM-F12 for 20–45 min. The trypsinization resulted in

detachment of an upper layer of cells containing all the astrocytes, whereas the microglia

remained attached to the bottom of the well. The medium containing detached cells was

removed and replaced with initial mixed glial-conditioned medium. Twenty-four hours

after trypsinization, the attached cells were subjected to the different treatments.

2.3. Primary culture of astrocytes Astrocytes were isolated from 2-day-old rats as previously described (Blondeau

et al., 1993), with minor modifications (Silva et al., 1999). Cells were resuspended in

DMEM containing 11 mM sodium bicarbonate, 71 mM glucose and 1% antibiotic

antimycotic solution supplemented with 10% FBS, plated (2.0 X 105 cells/cm2) on 75 cm2

culture flasks and cultured for 10 DIV.


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2.4. Primary neuronal cell cultures Neurons were isolated from foetuses of 17–18-day pregnant Wistar rats, as

described previously (Silva et al., 2002). Pregnant rats were anesthetized and

decapitated. The foetuses were collected in HBSS and rapidly decapitated, the brain

cortex was mechanically fragmented, and the fragments transferred to a 0.5 g/L trypsin

in Ca2+ and Mg2+ free HBSS medium and incubated for 15 min at 37ºC. After

trypsinization, cells were washed twice in calcium and magnesium free HBSS containing

10% FBS, and resuspended in Neurobasal medium supplemented with 0.5 mM L-

glutamine, 25 µM L-glutamic acid, 2% B-27 Supplement, and 0.12 mg/ml gentamicin.

Aliquots of 1 X 105 cells/cm2 were plated on poly-D-lysine coated 75 cm2 culture flasks

and maintained at 37ºC in a humidified atmosphere of 5% CO2. Every 3 days, 0.5 ml of

old medium was removed by aspiration and replaced by the same volume of fresh

medium without L-glutamic acid.

2.5. Neuron-microglia mixed cultures Primary cortical microglia were isolated from mixed glial cultures by mild

trypsinization as described above. Remaining microglia was removed by trypsinization

from the bottom of the plates and seeded on top of 2 DIV neurons at a density of 3.6 X

104 cells/cm2. Mixed neuron-microglia cultures were kept for 24 h before treatment with

UCB.

2.6. Cell treatment Microglial cells were incubated in the absence (control) or in the presence of 50

µM UCB plus 100 µM HSA, for 4 h or 24 h, at 37ºC. A UCB stock solution (10 mM) was

prepared in 0.1 M NaOH immediately before use and the pH of the incubation medium

was restored to 7.4 by addition of equal amounts of 0.1 M HCl. All the experiments with

UCB were performed under light protection to avoid photodegradation.

To study the role of soluble factors released by astrocytes or neurons upon UCB

exposure, microglial cells were incubated for 4 or 24 h with conditioned medium from

either 10 DIV astrocytes or 8 DIV neurons prior exposed to the same UCB and HSA

levels for 12 h. Controls were performed in the absence of UCB. Neuron-microglia mixed

cultures at 3 DIV were incubated in the absence (control) or in the presence of 50 µM

UCB plus 100 µM HSA, for 24 h, at 37ºC.

Neuron-microglia mixed cultures at 3 DIV were incubated in the absence (control)

or in the presence of 50 µM UCB plus 100 µM HSA, for 24 h, at 37ºC.

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2.7. Measurement of cytokine release Aliquots of the cell culture media were collected at the end of the incubations and,

after removal of cellular debris by short centrifugation, placed in a 96-well microplate and

assessed in triplicate for TNF-α, IL-1β and IL-6 with specific DuoSet® ELISA

Development kits from R&D Systems, according to the manufacturer’s instructions.

Results were expressed as fold change vs. control.

2.8. Gelatin zymography Aliquots of culture supernatant were analyzed by sodium dodecyl sulphate-

polyacrylamide gel electrophoresis (SDS-PAGE) zymography in 0.1% gelatine–10%

acrylamide gels under non-reducing conditions. After electrophoresis, gels were washed

for 1 h with 2.5% Triton X-100 (in 50 mM Tris pH7.4; 5 mM CaCl2; 1μM ZnCl2) to remove

SDS and renature the MMP species in the gel. Then the gels were incubated in the

developing buffer (50 mM Tris pH7.4; 5 mM CaCl2; 1μM ZnCl2) overnight to induce

gelatine lysis. For enzyme activity analysis, the gels were stained with 0.5% Coomassie

Brilliant Blue R-250 and destained in 30% ethanol/10% acetic acid/H2O. Gelatinase

activity, detected as a white band on a blue background, was quantified by computerized

image analysis and normalized for total cellular protein.

2.9. Assessment of microglial phagocytic properties After treatment with UCB, cells were incubated with 0.0025% (w/w) 1 μm

fluorescent latex beads for 75 min at 37ºC and fixed with freshly prepared 4% (w/v)

paraformaldehyde in phosphate-buffered saline (PBS). Microglia were counterstained

with a primary antibody raised against Iba-1 (goat, 1:500) followed by a secondary Alexa

Fluor 594 chicken anti-goat antibody (1:200). Green- and red-fluorescence images of

fifteen random microscopic fields (original magnification: 630X) were acquired per

sample. The number of ingested beads per cell was counted in approximately 250 cells

per sample.

2.10. Quantification of nitrite levels

Nitric oxide levels were estimated by measuring the concentrations of nitrites,

which are the resulting NO metabolites. Briefly, supernatants free from cellular debris

were mixed with Griess reagent in 96-well tissue culture plates for 10 min at room

temperature in the dark. The absorbance at 540 nm was determined using a microplate

reader (Bio-Rad Laboratories, Hercules, CA, USA).


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2.11. Evaluation of cell death Necrotic-like cell death was estimated by evaluating the activity of LDH released

by nonviable cells, as well as by monitoring the cellular uptake of the fluorescent dye PI

[3,8-diamino-5-(3-(diethylmethylamino)propyl)-6-phenyl phenanthridinium diiodide].

LDH was determined in the incubation medium using a cytotoxicity detection kit

as described previously (Silva et al., 2002). The reaction was performed in a 96-well

microplate and the absorbance measured at 490 nm. All readings were corrected for the

possible interference of UCB absorption. The maximum amount of releasable LDH was

obtained by lysing non-incubated cells with 2.0% Triton X-100 in DMEM for 5 min and

the results were expressed as fold change vs. control.

PI readily enters and stains non-viable cells, but cannot cross the membrane of

viable cells. This dye binds to double-stranded DNA and emits red fluorescence (630

nm; absorbance 493 nm). Unpermeabilized adherent cells cultured on coverslips were

incubated with a 75 μM PI solution for 15 min in the absence of light. Subsequently, cells

were fixed with freshly prepared 4% (w/v) paraformaldehyde in PBS and the nuclei

stained with Hoechst 33258 dye. Red-fluorescence and U.V. images of ten random

microscopic fields (original magnification: 400X) were acquired per sample and the

percentage of PI positive cells was counted and expressed as fold vs. respective control.

2.12. Neurite Extension and Ramification Neurite extension and ramification were assessed by the immunofluorescence

detection of the cytoskeletal protein MAP-2, as described previously (Falcão et al.,

2007). Briefly, cells were fixed as described above and a standard indirect

immunocytochemical technique was carried out using a mouse anti-MAP-2 antibody

(1:100) as the primary antibody and a horse FITC-labeled anti-mouse antibody (1:227)

as the secondary antibody.

In neuron-microglia mixed cultures, microglia were counterstained with a primary

antibody raised against Iba-1 (goat, 1:500) followed by a secondary Alexa Fluor 594

chicken anti-goat antibody (1:200) and the nuclei stained with Hoechst 33258 dye.

Fluorescence was visualized using a Leica DFC490 camera adapted to an

AxioSkope® microscope. Green- and red-fluorescence images of ten random

microscopic fields (original magnification: 400X) were acquired per sample. Evaluation of

neurite extension and number of nodes from individual neurons, were traced manually

using ImageJ software (National Institutes of Health).

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2.13. Statistical Analysis Results of at least three different experiments, carried out in duplicate, were

expressed as mean ± SEM. Differences between groups were determined by one-way

ANOVA with Dunnett’s or Bonferroni’s multiple comparisons post tests, using Instat 3.05

(GraphPad Software, San Diego, CA). Statistical significance was considered for a p

value less than 0.05.

3. Results

3.1. Conditioned media from UCB-treated astrocytes and neurons modulate microglial secretion of cytokines, as compared to UCB-activated microglia

Microglial cells are important sources of cytokines in the brain (Hanisch, 2002). In

fact, a recent report from our group demonstrated that microglia, upon UCB stimulation,

engages an inflammatory reaction which entails IL-1β IL-6 and TNF-α production and

follows different temporal profiles of secretion (Silva et al., 2010). However, the influence

of neighbouring cells must be considered when evaluating the response of microglia to

toxic molecules like UCB. Indeed, several authors have described that neurons or

astrocytes can modulate the inflammatory reaction undertaken by microglia (Giulian et

al., 1993; Min et al., 2006; Mott et al., 2004; Polazzi and Contestabile, 2006). For that

matter we compared cytokine secretion by microglia directly exposed to UCB or to

astrocytes and neuron conditioned media (ACM and NCM, respectively) after incubation

with UCB. Our results indicated that both UCB-treated ACM and NCM completely

counteracted (p<0.01) the UCB-induced increase in IL-1β (1.5 fold, p<0.01), as depicted

in Figure III.1A. Regarding TNF-α production (Figure III.1B), only NCM was able to

significantly prevent (p<0.05) the elevation elicited by UCB (1.5 fold, p<0.05). Curiously,

both ACM and NCM obtained after treatment with UCB led to an IL-6 overproduction by

microglia (2.7 and 2.8-fold, respectively; p<0.01 vs. control) which proved to be

significantly higher than the one triggered by the direct effect of UCB in microglia (p<0,01

for both UCB-treated ACM and NCM), as can be seen in Figure III.1C. Therefore, these

findings indicate that UCB-stimulated astrocytes and neurons dampen microglial

inflammatory response triggered by UCB in isolated cells, since the release of pro-

inflammatory mediators such as IL-1β and TNF-α is down-regulated while the secretion

of IL-6, which is considered to exert anti-inflammatory effects (Loddick et al., 1998), is

greatly enhanced.


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Fig. III.1. Conditioned media derived from astrocytes (ACM) and neurons (NCM) incubated with UCB modulate the secretion of IL-1β, TNF-α and IL-6 by microglia directly exposed to UCB. Rat cortical microglial cells were treated for 24 h with 50 µM UCB in the presence of 100 µM HSA, or with ACM or NCM from cells exposed to the same UCB and HSA levels for 12 h. Controls were performed in the absence of UCB. IL-1β, TNF-α and IL-6 concentrations in the media were determined by ELISA and expressed as mean ± SEM fold change vs. control from three independent experiments performed in triplicate. *p<0.05 and **p<0.01 vs. control; §p<0.05 and §§p<0.01 vs. UCB alone.

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3.2. Conditioned media from UCB-treated astrocytes and neurons have opposing effects on microglial MMP-9 activity, as compared to UCB-activated microglia UCB has been previously shown to enhance the activity of MMP-9 and -2,

proteases that actively participate in inflammatory events. Moreover, the same study also

evidenced the existence of regulatory interactions between cytokine secretion and MMP

activation upon UCB exposure (Silva et al., 2010). Since conditioned media from UCB-

exposed astrocytes and neurons showed to modulate the secretion of inflammatory

mediators, we wondered what effect could ACM and NCM exert on MMP activation. The

results show that UCB-treated NCM and direct UCB stimulation have similar activation

levels of MMP-2 (p<0.05 vs. control) but ACM from astrocytes exposed to UCB did not

produce any significant effect (Figure III.2A). Curiously, our findings evidenced opposing

effects of ACM and NCM upon UCB stimulation on MMP-9 activity by microglia directly

exposed to UCB (Figure III.2B). While ACM abrogated (p<0.05) the increase in MMP-9

activity elicited by UCB in microglia (1.1-fold, p<0.05), NCM further aggravated such

effect (1.2-fold; p<0.01 vs. control and p<0.05 vs. UCB alone).

3.3. Nitric oxide (NO) release by microglia is elicited by UCB and is higher by naïve microglia exposed to conditioned medium from UCB-treated neurons Microglia and astrocytes generate an NO burst in response to injury, which may

be cytotoxic (Cassina et al., 2002; Kawase et al., 1996) and trigger the clearance of

damaged cells from the CNS (Bal-Price and Brown, 2001; Golde et al., 2002). Although

UCB has been shown to elicit NO release in neurons (Brito et al., 2010), no evidence

was ever confirmed regarding microglial cells exposed to UCB. Therefore, this study is

the first to demonstrate that UCB can induce the generation of NO by microglia (1.3-fold;

p<0.05 vs. control). Moreover, the results displayed in Figure III.3 indicate that the

evoked NO production is even more enhanced when non-treated (naïve) microglia are

exposed to conditioned medium derived from neurons incubated with UCB, NCM, (1.5-

fold; p<0.01 vs. control and p<0.05 vs. UCB alone). On the other hand, conditioned

medium from astrocytes exposed to UCB, ACM, did not significantly alter the amount of

NO release by UCB-stimulated microglia (1.3-fold, p<0.05 vs. control but N.S. vs. UCB

alone). Data show that neighbouring neurons may further increase the nitrosative stress

induced by UCB in microglia.


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Fig. III.2. MMP-9 activation in microglia directly exposed to UCB is prevented by conditioned medium derived from UCB-treated astrocytes (ACM), but enhanced by conditioned medium derived from UCB-treated neurons (NCM). Culture supernatants from rat cortical microglial cells were harvested after 24 h incubation with 50 µM UCB in the presence of 100 µM HSA or with ACM or NCM from cells exposed to the same UCB and HSA levels for 12 h. Controls were performed in the absence of UCB. Samples were subjected to zymography as described in Methods. (A) Representative gels of one experiment are reported. MMP-2 and MMP-9 were identified by their apparent molecular mass of 67 and 92 kDa, respectively. (B ,C) Intensity of the bands was quantified by scanning densitometry, standardized with respect to total protein content and expressed as mean ± SEM fold change compared with those obtained in control conditions. *p<0.05 and **p<0.01 vs. respective control; §p<0.05 vs. UCB alone.

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Fig. III.3. NO release by UCB-activated microglia is intensified by conditioned medium derived from UCB-treated neurons (NCM), but not changed by conditioned medium derived from UCB-treated astrocytes (ACM). Rat cortical microglial cells were treated for 24 h with 50 µM UCB in the presence of 100 µM HSA or with ACM or NCM from cells exposed to the same UCB and HSA levels for 12 h. Controls were performed in the absence of UCB. NO production was estimated by the quantification of nitrite levels and expressed as mean ± SEM fold change from three independent experiments performed in duplicate. *p<0.05 and **p<0.01 vs. control and §p<0.05 vs. UCB alone.

3.4. Loss of viability in UCB-stimulated microglia is less in naïve microglia exposed to conditioned medium from UCB-treated astrocytes (ACM), but higher if treated with medium from neurons incubated with UCB (NCM) To evaluate whether the loss of microglia viability upon exposure to ACM and

NCM was different from the one produced by the direct action of UCB on microglial cell

death, we assessed the extracellular accumulation of LDH, a characteristic feature of cell

membrane integrity loss. Our results evidenced that ACM does not produce any change

over the results observed in the absence of UCB (Figure III.4) and was significantly less

(p<0.01) than the release of LDH obtained in UCB-stimulated microglia (1.5 fold,

p<0.01). In contrast, the values achieved with NCM-treated microglia were clearly higher

(1.8 fold, p<0.01 vs. control and p<0.05 vs. UCB alone). This finding strengthens again

the notion that neurons may further damage reactive microglia increasing UCB injury in

brain parenchyma.


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Fig. III.4. LDH leakage by UCB-activated microglia is completely abrogated by conditioned medium derived from UCB-treated astrocytes (ACM) but enhanced by conditioned medium derived from UCB-treated neurons (NCM). Culture supernatants from rat cortical microglial cells were harvested after 24 h incubation with 50 µM UCB in the presence of 100 µM HSA or with ACM or NCM from cells exposed to the same UCB and HSA levels for 12 h. Controls were performed in the absence of UCB. Samples were used for determination of LDH activity. Results are mean ± SEM from three independent experiments performed in duplicate. **p<0.01 vs. control; §p<0.05 and §§p<0.01 vs. UCB alone.

3.5. Microglial phagocytic properties enhanced by UCB further increase by medium collected from neurons exposed to UCB Recently we have described that microglia reacts to UCB by the acquisition of an

early phagocytic phenotype that afterwards shifted to a rather inflammatory state (Silva

et al., 2010). Since icteric NCM was shown to decrease the production of pro-

inflammatory cytokines, but to promote microglia demise, we wondered how microglia

would react if surrounded by neurons in an icteric parenchyma. Interestingly, as can be

seen in Figure III.5, the uptake of fluorescent latex beads by microglia is stimulated in the

presence of UCB, but even more by the icteric NCM (~40%, p<0.05). Therefore, UCB

seems to induce the release of soluble factors from neurons that stimulate the

phagocytic role of microglia. Whether deleterious consequences occur either by an

increased time of exposure to UCB or by higher UCB levels, are to be seen in future

studies.

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Fig. III.5. Microglia phagocytic ability increases upon exposure to UCB, and this property is enhanced by icteric NCM derived from UCB-treated neurons. Rat cortical microglial cells were treated for 24 h with 50 µM UCB in the presence of 100 µM HSA or with ACM or NCM from cells exposed to the same UCB and HSA levels for 12 h. Controls were performed in the absence of UCB. Cells were incubated with 1 μm green fluorescent latex beads as described in Methods. (A) Microglial cells were counterstained with an antibody raised against Iba-1 (red) Representative results of one experiment are shown. Scale bar 20 μm. (B) Results are expressed as number of ingested beads per cell (± SEM) from three independent experiments. *p<0.05 and **p<0.01 vs. control; §p<0.05 and §§p<0.01 vs. UCB alone.


95

3.6. UCB-induced neuronal network impairment is prevented by microglia environment

Considering that neurons showed to greatly influence microglial reactivity using a

conditioned medium model and that the phagocytic profile seems to be promoted under

these conditions, we decided to invert the picture and take a look at the effects of UCB-

reactive microglia on neurons. For that matter we used a neuron-microglia mixed culture

system where cell-to-cell contact could take place. Since immature cortical neurons have

previously been shown to be more vulnerable than the most differentiated ones to UCB-

induced cell death, glutamate release and TNF-α secretion (Falcão et al., 2006), we

chose to explore the effect of microglia on 3 DIV neurons, a differentiation stage where

UCB seems to produce a more injurious outcome.

As previously reported by Falcão et al. (2007) 24 h exposure of immature cortical

neurons to UCB decreases neuronal survival and causes an impairment of neurite

development. The involvement of microglia in neurite outgrowth during brain

development has been acknowledged (Cuadros and Navascues, 1998), as well as in

pathologic events (Munch et al., 2003; Rozenfeld et al., 2003). Activated microglia has

been accounted for as either neurotoxic (Zhang and Fedoroff, 1996) or neuroprotective

in several models (Nakajima et al., 2007) due to its inflammatory and phagocytic nature.

Nonetheless, the impact of microglia on neuronal plasticity in an icteric milieu had not

been previously evaluated. Hence, we decided to verify if the UCB-elicited network

impairment in neurons is modulated by the presence of microglia when mixed cultures of

both cell types are used. As represented in Figure III.6, microglia when cultivated with

neurons are able to protect the injury caused by UCB at the level of neurite extension

(p<0.01) and ramification (p<0.05).

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Fig. III.6. Reduction of neurite extension and ramification by UCB in immature neurons is counteracted by the presence of microglia. Cortical neurons and neuron-microglia mixed cultures at 3 DIV were exposed for 24 h, to either no addition (control), or to 50 µM UCB as described in Methods. Neurite extension (A) and number of nodes (B) were identified by immunolabeling for MAP-2 (green), quantified by ImageJ and expressed as arbitrary units ± SEM. Microglial cells were counterstained with an antibody raised against Iba-1 (red). Representative results of one experiment are shown in (C). **p<0.01 vs. control; §p<0.05 and §§p<0.01 vs. UCB alone. Scale bar 40 µm.

Therefore, defensive properties on the neuronal injury by UCB seem to be a key

role of microglia when UCB gets into the brain. We hypothesize that the additional

presence of risk factors such as sepsis or prematurity may change the protective role of

microglia. We also questioned if the effects observed were really a protection or resulted

from the increased microglia phagocytosis previously observed. Thus we next

investigated the influence of microglia in neuron viability upon UCB exposure.


97

3.7. Neuronal cell death triggered by UCB is diminished in the presence of microglia Interplay between neurons and microglia is widely recognized (Biber et al., 2007;

Zhang and Fedoroff, 1996). In fact, several reports have demonstrated that activated

microglia can decrease neuronal viability (Gibbons and Dragunow, 2006) or, by the other

hand, exert a neuroprotective role (Figueiredo et al., 2008; Nakajima et al., 2007).

Indeed, as demonstrated in Figure III.7, the propensity of microglia to decrease cellular

necrosis is reinforced in the presence of UCB, with a completely abrogated UCB-induced

death of immature neurons (p<0.01) in mixed cultures of neurons and microglia.

Fig. III.7. Decreased neuronal survival by UCB disappears when microglia is nearby. Cortical neurons or neuron-microglia mixed cultures at 3 DIV were exposed for 24 h, to either no addition (control), or 50 µM UCB and further incubated with 75 μM PI as described in Methods. Results are percentage of PI-positive cells and are expressed as mean ± SEM from three independent experiments performed in triplicate **p<0.01 vs. respective control and §§p<0.01 vs. UCB alone.

These results reinforce the neuroprotective role that microglia may exert in

preventing UCB neurological damage. However, given the acknowledged phagocytic

nature of microglia and the fact that icteric NCM further enhances that property, we

believe that this apparent protection results from the phagocytic clearance of UCB-

injured neurons, similarly to the events described in other disease models (Napoli and

Neumann, 2009; Neumann et al., 2009; Tanaka et al., 2009).

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4. Discussion

Cell-to-cell interactions as well as the release of soluble mediators are essential

regulators of many critical functions in brain health and disease. In this study we

demonstrated, for the first time, that astrocytes and neurons stimulated by UCB can

modulate microglial reactivity through different pathways. UCB-treated astrocytes lead to

a decrease in the inflammatory response elicited by UCB in microglia and ultimately

prevent UCB-induced cell demise. On the other hand, although UCB-exposed neurons

release soluble factors with the capacity to down-regulate microglial production of pro-

inflammatory cytokines, neurons also lead to increased NO production and further

aggravate UCB-induced MMP-9 activity in microglia. Interestingly, neurons exacerbate

microglial early phagocytic phenotype while also increase microglia demise by UCB. Our

study is the first to evaluate if injurious effects of UCB on neurons are modified in the

presence of microglial cells, thus taking into consideration probable cellular interactions.

Curiously, our findings show that microglial cells counteract the deleterious effects

produced by UCB on neuronal cell death and neurite extension and ramification.

Altogether, these observations allow us to hypothesize that UCB-evoked injury in

neurons is signaling microglia to engage phagocytic clearance in an attempt to limit

injury extension, rather than exacerbating inflammation.

The contribution of microglial immune response to either damage or repair is a

broadly discussed subject (Amor et al., 2010). Additionally, the influence of neighboring

cells on microglial reactivity is renowned, substantiating the observed immunomodulatory

effects produced by icteric ACM and NCM. In fact, when evaluating the role of microglia

in organometallic compounds’ toxicity, astrocytes were shown to dampen TNF-α

production and, additionally, were not able to induce morphogical changes (Eskes et al.,

2003). Furthermore, astrocytes can attenuate microglial activation in several disease

models, thus preventing neurodegeneration (Giulian et al., 1993; Rozenfeld et al., 2003).

It is also interesting to notice that, in our work model, UCB-treated astrocytes and

neurons greatly enhanced IL-6 production by microglia. Similar effects were observed in

studies regarding microglial reactivity to methylmercury where interaction between

activated microglia and astrocytes was shown to increase local IL-6 release, which may

cause astrocyte reactivity and ultimately culminate in neuroprotection (Eskes et al.,

2002).

We have recently demonstrated that UCB-induced cytokine release by microglia,

particularly IL-1β, originate MMP-9 and -2 activation (Silva et al., 2010). Thus, the

observed decrease in microglial MMP-9 activity after exposure to icteric ACM could be

explained by the regulatory interactions between cytokines and MMPs, since the


99

production of the formers is also hampered in this particular case. Collectively, our

findings suggest an overall protective effect exerted by astrocytes on microglia, upon

UCB stimulation. We theorize that microglia, when isolated, respond intensively to UCB

stimulation, but previously activated astrocytes drive microglia to refrain its response.

Other authors have postulated that this is a feasible mechanism to prevent excessive

brain inflammation (Min et al., 2006).

In this work we also describe the down-regulatory effect of icteric NCM upon

UCB-stimulated microglial secretion of pro-inflammatory cytokines, particularly IL-1β and

TNF-α. Accordingly, neurons reduce glial secretion of TNF-α after treatment with the

bacterial endotoxin lipopolysaccharide (LPS) emphasizing their role as immune

responsiveness regulators (Chang et al., 2001; Mott et al., 2004). Similarly, neurons

constitutively release factors that maintain microglia in a quiescent state in the healthy

brain (Biber et al., 2007), such as the protein CD200, a molecule shown to down-

regulate microglial inflammatory response (Broderick et al., 2002; Deckert et al., 2006).

Nevertheless, under inflammatory or deleterious conditions, microglia’s behavior might

be modified, as suggested by the study of Hasegawa et al. (2007) where the contact rate

between activated microglia and hippocampal pyramidal cells’ dendrites is increased.

Additionally, the severity of neuronal injury has been reported to determine the release of

trophic or pro-inflammatory mediators form microglia (Lai and Todd, 2008). For example,

interaction of microglia with apoptotic neurons leads to a progressive down-regulation of

pro-inflammatory microglial functions (Minghetti et al., 2005). Considering that neurons

reveal higher susceptibility to cell death than astrocytes, in the presence of high UCB

levels (Brites et al., 2009; Silva et al., 2002), we postulate that this event leads to the

release of factors by neurons which give the impression to influence microglial response

to UCB in a more marked fashion than those produced by astrocytes. In fact, our results

reveal that conditioned medium derived from UCB-stimulated neurons dampens

microglial production of pro-inflammatory cytokines, yet increases MMP-9 activity and

NO secretion, culminating in microglial death. These observations led to the assumption

that UCB-injured neurons might be signaling microglia to shift its activity state rather than

exerting a down- or up-regulation of its functions. Indeed, a previous report regarding

microglial reactivity to UCB has already identified two different phenotypes (phagocytic

and inflammatory) which occurred in an alternate fashion possibly indicating the

existence of two different microglial subpopulations reacting towards UCB (Silva et al.,

2010). In the present study we observed that soluble factors released by UCB-damaged

neurons are able to enhance the microglial phagocytic ability when compared to the one

showed by UCB-stimulated microglia. These data reinforce the belief that neuron-

microglia interaction modifies the response phenotype of the latter cells, upon UCB

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exposure. Consistently, other authors have reported that microglia recognize and

phagocyte dying neurons (Petersen and Dailey, 2004; Witting et al., 2000) providing a

clearance of potentially harmful debris.

Although MMP-9 activation and increase in NO production are normally included

in the inflammatory reaction engaged by microglia, in this case we may consider that

these events can be related to the acquisition of a phagocytic profile. In fact, NO has

been proven to regulate phagocytosis (Kopec and Carroll, 2000) and to trigger the

clearance of damaged cells (Cassina et al., 2002; Kawase et al., 1996). Furthermore,

MMPs can be involved in chemoattraction of microglial cells to injured sites since they

participate in the cleavage of fractalkine from neuronal membranes following toxic insults

(Chapman et al., 2000). MMPs also have the ability to degrade the basal lamina

surrounding the blood-brain barrier allowing infiltration of immune cells (Michaluk and

Kaczmarek, 2007; Rosenberg, 2002) consequently participating in phagocytic events

(Buss et al., 2007).

Nevertheless, the engagement of such a dramatic phenotype such as microglial

phagocytosis may culminate in cell death. Our observations regarding the influence of

UCB-reactive neurons on microglial response seem to corroborate this concept since cell

death is further aggravated under these conditions. Other studies have also reported that

NCM produces an overactivation of LPS-stimulated microglia culminating in apoptotic

elimination as a safety mechanism (Polazzi and Contestabile, 2006).

Besides studying the influence of soluble factors released by astrocytes or

neurons stimulated by UCB on microglial response, we intended to advance one step

further in the understanding of UCB-induced neurotoxicity and add neuron-microglia cell-

to-cell interactions to the puzzle. For this matter we used neuron-microglia mixed

cultures to study the effects of UCB on network dynamics and cell death, since isolated

immature neurons proved to be more affected by UCB than more differentiated cells in

these particular features (Falcão et al., 2007). Unexpectedly, microglia proved to

completely counteract UCB-elicited neurite impairment and loss of viability. Other

authors have proven opposite results concerning the effects of microglia on neurite

outgrowth and cell viability. For instance, Munch et al. (2003) demonstrated that

microglial activation induces cell death and inhibits neurite outgrowth in neuroblastoma

cells. Microglia is also involved in neuron loss in Alzheimer´s disease (Fuhrmann et al.,

2010) and may cause neuronal cell death and degeneration (Gibbons and Dragunow,

2006; Rozenfeld et al., 2003). Taking into account all these confounding aspects, we

hypothesize that this apparent microglial protection of UCB-induced neurotoxicity may

indeed be regarded as a neuroprotective measure aiming at limiting injury extension by

the phagocytic clearance of damaged neurons. Phagocytosis of amyloid deposits or


101

cellular debris by microglia was also proven to be important in Alzheimer´s disease

(Simard et al., 2006) and multiple sclerosis (Takahashi et al., 2007). Thus, clearance of

tissue debris performed by microglia following injury might constitute a regenerative

measure (Napoli and Neumann, 2009; Neumann et al., 2009).

In summary, our findings indicate that astrocytes activated by UCB release

soluble factors that reduce microglial inflammatory reaction, probably in an attempt to

refrain deleterious overactivation. On the other hand, UCB-stimulated neurons down-

regulate microglial production of inflammatory cytokines but further increase its

phagocytic abilities and ultimately lead to microglial elimination. Furthermore, microglia

counteract UCB-evoked impairment of neurite extension and ramification as well as

neuronal cell death in a mixed culture model. Hence, we hypothesize that UCB-injured

neurons might be signaling microglia to engage a phagocytic phenotype while hampering

inflammatory reaction in an attempt to constrain lesion extent. In conclusion, this study

introduces important knowledge to the understanding of the molecular basis of UCB-

induced neurotoxicity as it adds, for the first time, an important piece to the puzzle, e.g.

cellular interactions between microglia and other nerve cells in a model mimicking acute

neonatal jaundice.

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Chapter IV

UNCONJUGATED BILIRUBIN NEUROTOXICITY IS MODULATED BY MICROGLIA AND PREVENTED BY

GLYCOURSODEOXYCHOLIC ACID AND INTERLEUKIN-10

Sandra L. Silva,1 Ana R. Vaz,1 Maria J. Diógenes,2,3 Nico van Rooijen,4 Ana M. Sebastião,2,3 Adelaide Fernandes,1 Rui F. M. Silva,1 Dora Brites1

1Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL),

Faculdade de Farmácia, University of Lisbon, Av. Professor Gama Pinto, Lisbon

1649-003, Portugal

2Institute of Pharmacology and Neurosciences, Faculty of Medicine University of Lisbon,

Lisbon, Portugal. Av. Professor Egas Moniz, 1649-028 Lisbon, Portugal

3Unit of Neurosciences, Institute of Molecular Medicine, University of Lisbon, Portugal.

Av. Professor Egas Moniz, 1649-028 Lisbon, Portugal

4Department of Molecular Cell Biology, Faculty of Medicine, Amsterdam, The

Netherlands. Van der Boechorststraat 7, 1081 BT, Amsterdam, The Netherlands

Acta Neuropathologica, 2010 (submitted)

106

Acknowledgments The authors would like to thank Prof. Helmut Kettenmann’s research group for sharing

their expertise on microglia, Liliana Bernardino for her skill with organotypic-cultured

hippocampal slices and Pedro Pereira and Ana Isabel Pereira for technical assistance.

This work was supported by PTDC/SAU-NEU/64385/2006 grant, from Fundação para a

Ciência e a Tecnologia, Lisbon, Portugal (to D.B.). S.L.S. was recipient of a PhD

fellowship (SFRH/BD/30326/2006) from FCT.

Microglia, glutamate and nitric oxide as key players in hyperbilirubinemia

107

Abstract Microglia has recently emerged as a crucial mediator of CNS inflammation since

they are exquisitely sensitive to brain injury. The role of microglia during neonatal

jaundice is unrecognized, although we evidenced that microglia reacts to unconjugated

bilirubin (UCB) by engaging a phagocytic phenotype followed by the secretion of

cytokines and glutamate. In addition, UCB injury to neurons involve short- and long-term

alterations in neurite outgrowth and synaptic density, namely in hippocampal neurons,

and is mediated by oxidative and nitrosative stress, raising the possibility of having an

impact on infant’s learning.

This study investigated microglia neuroprotective or neurotoxic effects in a cell-to-

cell concerted action in response to UCB, namely in the production of glutamate and

nitric oxide (NO), using organotypic cultured-hippocampal slices. Involvement of

glutamate and NO in UCB-mediated toxicity to immature neurons was addressed using

MK-801 (a NMDA glutamate-subtype receptor antagonist) and L-NAME (a non-specific

NO synthase inhibitor). Therapeutic potential of glycoursodeoxycholic acid (GUDCA) and

interleukin (IL)-10, with antioxidant and immunossupressive properties, in UCB-induced

neurodegeneration was also evaluated.

Microglia revealed to participate in glutamate homeostasis and to induce the

release of this neurotransmitter and NO in UCB-treated hippocampal slices, which

showed to be mediators in neuritic impairment, and cell death. Either GUDCA or IL-10

counteracted these insidious effects on immature neurons, but only GUDCA showed

preventive effects on cell death, synaptic changes and release of glutamate and NO in

UCB-treated cortical neurons and hippocampal slices.

Collectively our data reveal microglia, glutamate and NO as key players in UCB-

induced neurotoxicity and point to GUDCA as a promising therapy in infant’s

hyperbilirubinemia.

Keywords: Glutamate-mediated neurotoxicity; Glycoursodeoxycholic acid; Interleukin-10;

Long-term disabilities; Microglia-neuron interactions; Neurite outgrowth impairment; Nitric

oxide; Organotypic-cultured hippocampal slices; Unconjugated bilirubin.

Chapter IV

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1. Introduction Microglial cells are the first to respond to neural insults (Morioka et al., 1991) and

its reactivity may be differentially modulated depending on microenvironmental signals

(Zhang and Fedoroff, 1996). Activated microglia promote neuronal survival in ischemic

lesion and facial nerve axotomy models (Moran and Graeber, 2004). Nevertheless,

microglia can also release molecules, which may be harmful in acute brain insults and

neurodegenerative diseases (Raivich et al., 1999).

Hyperbilirubinemia, a very common neonatal condition, characterized by

increased serum levels of unconjugated bilirubin (UCB) (Dennery et al., 2001), is

associated with minor neurologic dysfunction (Soorani-Lunsing et al., 2001) and

correlated with the emergence of long-term neurodevelopment disabilities (Dalman and

Cullberg, 1999; Miyaoka et al., 2000). Several toxic effects have been accounted for

UCB neurotoxicity such as cell death by both necrosis and apoptosis (Rodrigues et al.,

2002a; Silva et al., 2001) and neuronal oxidative injury (Brito et al., 2008b; Vaz et al.,

2010). UCB has also been shown to inhibit glutamate uptake prolonging its presence in

the synaptic cleft (Silva et al., 1999; Silva et al., 2002) and to enhance its secretion in

both astrocytes (Falcão et al., 2005; Fernandes et al., 2004) and microglia (Gordo et al.,

2006) enabling N-methyl-D-aspartic acid (NMDA)-mediated excitotoxicity (Brito et al.,

2010; Grojean et al., 2000; Grojean et al., 2001; McDonald et al., 1998).

Microglia is one of the brain’s major sources of reactive oxygen species (ROS)

and reactive nitrosative species (RNS). In fact, microglial activation upon UCB exposure

has been demonstrated to engender inflammation (Silva et al., 2010), glutamate

secretion (Gordo et al., 2006) and nitric oxide (NO) increased production (unpublished

results).

The involvement of glutamate in cell death (Barger and Basile, 2001; Barger et

al., 2007; Hahn et al., 1988; Lee et al., 2000; Liang et al., 2008; Takeuchi et al., 2008)

and neuritic outgrowth regulation (Hoffman et al., 1996; Lee et al., 2005; Mattson et al.,

1988; Monnerie et al., 2003) has been widely established. Intimately linked to

excitotoxicity is the generation of destructive free radicals, especially RNS (Ha et al.,

2010). These species are accountable for synapse and neuron injury, can exacerbate

excitotoxicity (Sunico et al., 2010) and are broadly implicated in neurodegenerative

diseases (Bishop and Anderson, 2005). In a very recent paper it was evidenced that

excitotoxicity plays a key role in UCB-induced oxidative damage to rat cortical mature

neurons (Brito et al., 2010).

Finally, UCB seems to produce neurodevelopmental deficits since it has

deleterious effects in neurogenesis, neuritogenesis and synaptogenesis (Falcão et al.,

2007; Fernandes et al., 2009). Indeed, early exposure to UCB leads to impairment of


109

neuronal development by reducing dendrite extension and ramification, as well as

dendritic spine formation and synapse establishment (Falcão et al., 2007). Moderate

concentrations of UCB inhibit the induction of long term potentiation (LTP) in the

hippocampus by short-term (Zhang et al., 2003) or prolonged exposure (Chang et al.,

2009), indicating that neonatal jaundice may have deleterious consequences in learning

and memory. In fact, it was observed that the more the history of jaundice was severe

the highest difficulty for learning was revealed (Weir and Millar, 1997).

Glycoursodeoxycholic acid (GUDCA) was proven to reduce cell death induced by

UCB in astrocytes (Fernandes et al., 2007) and neurons (Brito et al., 2008a; Vaz et al.,

2010), and to abrogate the UCB-induced oxidative damage in neurons (Brito et al.,

2008a; Vaz et al., 2010). Moreover, immunosuppressive properties of GUDCA and

interleukin (IL)-10 were demonstrated on the release of pro-inflammatory cytokines by

UCB-treated astrocytes (Fernandes et al., 2007). IL-10 has also been shown to reduce

neuronal degeneration after central nervous system (CNS) injury (Bachis et al., 2001;

Park et al., 2007). However, to the best of our knowledge, no evidence was established

regarding the effect of GUDCA and IL-10 in the prevention of UCB-induced neurite

impairment along neuronal maturation.

Therefore, this study aimed at evaluating whether the production of glutamate

and NO could be elicited by UCB in an ex-vivo model of hippocampal slice cultures and,

more importantly, if this effect could be ascribed to microglial cells. This culture model

maintains the cytoarchitecture of the tissue, allowing interaction of multiple cell types in

the brain, namely neurons, astrocytes and microglia (Cho et al., 2007). Furthermore, we

intended to clarify the role of glutamate and NO in UCB-induced neurotoxicity by using

MK-801 (a NMDA glutamate-subtype receptor antagonist) and L-NAME (a non-specific

NO synthase inhibitor), and to elucidate the protective effects of GUDCA and IL-10 in

both isolated immature neurons and hippocampal slice cultures.

Our studies clearly demonstrate a key role of NO and overactivation of NMDA

receptors in UCB-induced impairment of neuritic outgrowth and cell death. Microglia

revealed to directly participate in glutamate homeostasis and NO production.

Additionally, we show, for the first time, that GUDCA and IL-10 prevent UCB-induced

loss of cell viability and extracellular glutamate accumulation. Moreover, they completely

abrogate the deleterious effects produced by UCB in neuronal network dynamics in a

very sustained manner along cellular differentiation, indicating that both short- and long-

term UCB harmful consequences on CNS could be in this way counteracted.

Furthermore, our findings report that UCB is able to down-regulate the expression of pre-

synaptic proteins, adding on the detrimental effects of UCB on synaptic plasticity, and

that only GUDCA prove efficacy in preventing this injurious outcome.

Chapter IV

110

2. Material and Methods 2.1. Chemicals

Neurobasal medium, B-27 Supplement (50x), Hanks’ balanced salt solution

(HBSS), HBSS without Ca2+ and Mg2+, gentamicin (50 mg/mL), trypsin (0.5 g/L) and

Alexa Fluor 594 chicken anti-goat IgG were acquired from Invitrogen (Carlsbad, CA,

USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and L-

glutamine, were purchased from Biochrom AG (Berlin, Germany). Antibiotic antimycotic

solution (20X), human serum albumin (HSA; fraction V, fatty acid free), bovine serum

albumin (BSA), Hoechst 33258 dye, mouse anti-β-actin antibody, propidium iodide (PI),

N-1-naphthylethylenediamine, N-ω-nitro-L-arginine methyl ester (NAME), [(+)-5-methyl-

10,11-dihydro-5Hdibenzo[a,d]cyclohepten-5,10-imine maleate)] (MK-801) and DAPI

were purchased from Sigma Chemical Co. (St. Louis, MO). UCB was also obtained from

Sigma and purified according to the method of McDonagh and Assisi, (1972).

Mouse anti-microtubule associated protein (MAP)-2, mouse anti- synaptosomal-

associated protein (SNAP)-25 and mouse anti-synaptophysin antibodies were from

Chemicon (Temecula, CA) and fluorescein isothiocyanate (FITC)-labeled horse antibody

anti-mouse was acquired from Vector (Burlingame, CA). GUDCA (minimum 96% pure)

was obtained from Calbiochem, Darmstadt, Germany and recombinant rat IL-10 from

R&D Systems, Minneapolis, MN.

Nitrocellulose membrane and Hyperfilm ECL were from Amersham Biosciences

(Piscataway, NJ, USA). Horseradish peroxidase-labeled goat anti-rabbit IgG and anti-

mouse IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cell

lysis buffer and LumiGLO® were from Cell Signaling (Beverly, MA, USA). L-glutamic acid

kit was purchased from Roche Molecular Biochemicals (Manheim, Germany).



2.2. Organotypic-cultured hippocampal slices Animal care followed the recommendations of European Convention for the



experimental animals). The Institutional Animal Care and Use Committee approved all

animal procedures. Every effort was made to minimize the number of animals used and

their suffering.

Organotypic-cultured hippocampal slices were prepared from P8-P10 Wistar rat

brains, according to the interface culture method (Stoppini et al., 1991), as previously


111

described (Markovic et al., 2005; Synowitz et al., 2006). Briefly, mice were killed by

decapitation, their brains removed under sterile conditions and the two hippocampi

isolated and cut in 400 µm coronal sections using a McIlwain tissue chopper. The

hippocampal slices were then transferred onto the 0.4-μm polycarbonate membrane in

the upper chamber of a Transwell tissue insert (Falcon model 3090, Becton Dickinson,

Lincoln Park, NJ), which were placed into a six-well plate (Falcon model 3502, Becton

Dickinson). Thereafter, slices were incubated in 1 mL culture medium per well containing

DMEM supplemented with 10% heat-inactivated FBS (medium 1). After overnight

equilibration of the organotypic-cultured hippocampal slices in medium 1, this was

exchanged for cultivation medium (medium 2). Medium 2 (100 mL) contains 25 mL heat-

inactivated horse serum, 580 μL bicarbonate (7.5%), 2 mL of L-glutamine solution, 25

mL HBSS, 10 μL of insulin (10 mg/mL), 1.2% glucose, 80 μL vitamin C (1 mg/mL), 1 mL

antibiotic-antimycotic solution, and 500 μL of 1 mol/L Tris in DMEM. Medium was

changed every day.

2.3. Preparation of microglia-depleted organotypic-cultured hippocampal slices Microglia-depleted organotypic-cultured hippocampal slices were obtained by 24

h treatment with liposomes filled with clodronate as described in (Markovic et al., 2005).

Liposomes were obtained from GOT Therapeutics (Berlin, Germany) and from the

Department of Molecular Cell Biology of the Free University of Amsterdam. For the

preparation of clodronate-liposomes, 86 mg of phosphatidylcholine and 8 mg of

cholesterol were combined with 10 ml of a clodronate (0.7 M; a gift of Roche Diagnostics

GmbH, Mannheim, Germany) solution and sonicated gently. The resulting liposomes

were washed to eliminate free drug. All liposomes were passed through a 12 mm filter

immediately prior to use in order to eliminate large lipid aggregates (Van Rooijen and

Sanders, 1994).

2.4. Organotypic-cultured hippocampal slices treatment Slices were maintained in vitro for a minimum of 3 days prior to use, a period over

which tissues recover from experimental trauma caused by the isolation procedure

(Huuskonen et al., 2005).

A stock solution of purified UCB was prepared in 0.1 N NaOH immediately before

use and the pH of the incubation medium was restored to 7.4 by addition of equivalent

amounts of 0.1 N HCl. All the experiments with UCB were performed under light

protection to avoid photodegradation. Organotypic-cultured hippocampal slices were

incubated in the absence (control) or in the presence of 50 µM UCB plus 100 µM HSA,

Chapter IV

112

for 24 h, at 37ºC. When appropriate, slices were pre-incubated with 50 µM GUDCA (from

a 5 mM stock solution) 1 h prior to UCB addition.

2.5. Assessment of cell death in organotypic-cultured hippocampal slices Cell death in organotypic-cultured hippocampal slices was assessed by

monitoring the cellular uptake of the fluorescent dye PI [3,8-diamino-5-(3-

(diethylmethylamino)propyl)-6-phenyl phenanthridinium diiodide]. PI readily enters and

stains non-viable cells, but cannot cross the membrane of viable cells. This dye binds to

double-stranded DNA and emits red fluorescence (630 nm; absorbance 493 nm). After

UCB treatment, slices were exposed to 2 µg/mL PI for 2 h and fixed by immersion in 4%

paraformaldehyde for 30 min, cryoprotected in 30% sucrose and stored at -20ºC until

use. Slices were cut in 15 µm-thick sections and the nuclei were stained with DAPI

(2ug/mL). Cellular uptake of PI was recorded by fluorescence microscopy using a

rhodamine filter and a Leica DFC490 camera adapted to an AxioSkope® microscope.

The percentage of PI-positive cells was quantified using ImageJ software (National

Institutes of Health).

2.6. Quantification of nitrite levels Nitric oxide levels were estimated by measuring the concentrations of nitrites

(NO2-), which are the resulting NO metabolites. Briefly, supernatants free from cellular

debris were mixed with Griess reagent [1 part 1% (w/v) sulfanilamide in 5% H3PO4, 1

part 0.1% (w/v) N-1-naphthylethylenediamine (v/v)] in 96-well tissue culture plates for 10

min at room temperature in the dark. The absorbance at 540 nm was determined using a

microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

2.7. Primary neuronal cell cultures Neurons were isolated from foetuses of 17–18-day pregnant Wistar rats, as

described previously (Silva et al., 2002). Pregnant rats were anesthetized and

decapitated. The foetuses were collected in HBSS and rapidly decapitated, the brain

cortex was mechanically fragmented, and the fragments transferred to a 0.5 g/L trypsin

in Ca2+ and Mg2+ free HBSS medium and incubated for 15 min at 37ºC. After

trypsinization, cells were washed twice in calcium and magnesium free HBSS containing

10% FBS, and resuspended in Neurobasal medium supplemented with 0.5 mM L-

glutamine, 25 µM L-glutamic acid, 2% B-27 Supplement, and 0.12 mg/ml gentamicin.

Aliquots of 1 x 105 cells/cm2 were plated on poly-D-lysine coated 12-well tissue culture

plates and maintained at 37ºC in a humidified atmosphere of 5% CO2. Every 3 days, 0.5


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ml of old medium was removed by aspiration and replaced by the same volume of fresh

medium without L-glutamic acid.

2.8. Cell treatment A 100 mM stock solution of L-NAME, a competitive nitric oxide synthase (NOS)

inhibitor, and a 1 mM stock solution of MK-801, a NMDA receptor antagonist, were

prepared in phosphate-buffered saline (PBS), pH 7.4. A stock solution of purified UCB

was prepared as described above.

Neurons at 3 days in vitro (DIV) were incubated in the absence (control) or in the

presence of 50 µM UCB plus 100 µM HSA, for 24 hr, at 37ºC. When appropriate, cells

were pre-incubated with 50 µM GUDCA (from a 5 mM stock solution) or 10 ng/mL

recombinant rat IL-10 (from a 50 µg/mL stock solution), 1 h prior to UCB addition.

Parallel sets of experiments were performed where cells were incubated with UCB alone

or in combination with 100 μM NAME or with 1 μM MK-801.

At the end of the incubation period (considered as 4 DIV throughout the text) cell-

free medium was removed and attached cells were fixed for 30 min with freshly prepared

4% paraformaldehyde in PBS, for immunocytochemical studies. In another set of

experiments, incubation medium was removed and cells were additionally cultured until

18 DIV, to study the long-term effects of UCB exposure.

2.9. Neurite extension and ramification Neurite extension and ramification were assessed by the immunofluorescence

detection of the cytoskeletal protein MAP-2, as described previously (Falcão et al.,

2007). Briefly, cells were fixed as described above and a standard indirect

immunocytochemical technique was carried out using a mouse anti-MAP-2 antibody

(1:100) as the primary antibody and a horse FITC-labeled anti-mouse antibody (1:227)

as the secondary antibody.

Fluorescence was visualized using a Leica DFC490 camera adapted to an

AxioSkope® microscope. Green-fluorescence images of ten random microscopic fields

(original magnification: 400X) were acquired per sample. Evaluation of neurite extension

and number of nodes from individual neurons was achieved by manual tracing using

ImageJ software (National Institutes of Health).

2.10. Measurement of glutamate Glutamate content in incubation medium or in hippocampal slice homogenates

was determined by an adaptation of the L-glutamic acid kit (Roche), using a 10-fold

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volume reduction. The reaction was performed in a 96-well microplate and the

absorbance measured at 490 nm. A calibration curve was used for each assay. All

samples and standards were analyzed in duplicate and the mean value was used.

2.11. Evaluation of cell death Necrotic-like cell death was assessed by monitoring the cellular uptake of the

fluorescent dye PI. Unpermeabilized adherent cells cultured on coverslips were

incubated with a 75 μM PI solution for 15 min in the absence of light. Subsequently, cells

were fixed with freshly prepared 4% (w/v) paraformaldehyde in PBS and the nuclei

stained with Hoechst 33258 dye.

Red-fluorescence and U.V. images of ten random microscopic fields (original

magnification: 400X) were acquired per sample and the percentage of PI positive cells

was counted.

2.12. Western blot assay Crude synaptosomes were obtained by differential centrifugation as described in

Huttner et al. (1983). Total cell lysates were extracted from neurons using the Cell lysis

buffer® and centrifuged at 800 X g to separate P1 (nuclear and debris) and supernatant

(S1) fraction. S1 fraction was centrifuged at 9200 X g to separate P2 (membranous

fraction) and S2 (cytosolic fraction). Western blot assay was carried out as usual in our

lab (Fernandes et al., 2006). Briefly, crude synaptosomal fractions were separated on a

12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).

Following electrophoretic transfer onto a nitrocellulose membrane and blocking with 5%

milk solution, the blots were incubated with primary antibody overnight at 4ºC [anti-

SNAP-25 (1:1000), anti-synaptophysin (1:2000) in TBS, or anti-β-actin (1:10000) in 5%

(w/v) bovine serum albumin] and with horseradish peroxidase-labelled secondary

antibody [anti-mouse (1:5000) or anti-rabbit (1:5000)] for 1 h at room temperature.

Protein bands were detected by LumiGLO® and visualized by autoradiography with

Hyperfilm ECL.

2.13. Statistical Analysis Results of at least three different experiments, carried out in duplicate, were

expressed as mean ± SEM. Differences between groups were determined by one-way

ANOVA with Dunnett’s or Bonferroni’s multiple comparisons post tests, using Instat 3.05

(GraphPad Software, San Diego, CA). P <0.05 was accepted as statistically significant.


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3. Results

3.1. Microglia modulate UCB-induced glutamate release and NO production in organotypic-cultured hippocampal slices Activated microglia can produce a plethora of harmful products such as glutamate

and NO in several disease models. Thus, we decided to evaluate glutamate and NO

production in organotypic-cultured hippocampal slices upon UCB exposure and to

determine if this production could be ascribed to microglia by the selective depletion of

these cells.In homogenates prepared from hippocampal slices incubated with 50 μM

UCB for 24 h, there was a significant increase in the amount of glutamate (3.8 fold,

p<0.01, Figure IV.1A) when compared to slices not incubated with UCB. Interestingly, in

microglia-depleted slices, the increase in glutamate induced by UCB was totally

prevented (p<0.01), suggesting that glutamate accumulation was mainly due to resident

microglia. The glutamate levels in the incubation media of UCB-treated slices (Fig.

IV.1B) were also enhanced (1.4 fold, p<0.05, Figure IV.1B), as compared with non-

treated slices, and the increase was even more pronounced in microglia-depleted slices

(p<0.01). These findings suggest that microglia affects glutamate homeostasis leading to

the accumulation of the neurotransmitter upon UCB exposure.

Fig. IV.1. Microglia modulate UCB-induced glutamate release in organotypic-cultured hippocampal slices. Hippocampal slices non-depleted or depleted in microglia, cultured for 3 days in vitro (DIV), were exposed for 24 h, to either no addition (control), or 50 µM UCB. Tissue concentrations of glutamate (A) or glutamate levels in incubation media (B) were evaluated using a colorimetric assay and expressed as mean ± SEM fold change compared with non-depleted in microglia, in the absence of UCB, from three independent experiments performed in duplicate. *p<0.05 and **p<0.01 vs. non-depleted and depleted in microglia, in the absence of UCB; §p<0.05 and §§p<0.01 vs. UCB in non-depleted in microglia.

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Furthermore, incubation of hippocampal slices with UCB also led to increased NO

release into the culture medium (1.4 fold, p<0.05), but not in microglia-depleted slice

cultures (p<0.05 vs. non-depleted slices) as depicted in Figure IV.2.

Fig. IV.2. Microglia modulate UCB-induced NO production in organotypic-cultured hippocampal slices. Hippocampal slices non-depleted or depleted in microglia, cultured for 3 days in vitro (DIV), were exposed for 24 h, to either no addition or 50 µM UCB. NO production was estimated by the quantification of nitrite levels and expressed as mean ± SEM from three independent experiments performed in duplicate. *p<0.05 vs. non-depleted and depleted in microglia, in the absence of UCB; §p<0.05 vs. UCB in non-depleted in microglia.

Since glutamate and NO have already been implicated as mediators in UCB-

neurotoxicity (Brito et al., 2010; Grojean et al., 2000; McDonald et al., 1998; Vaz et al.,

2010), as well as in other diseases, these findings suggest that microglial reactivity may

be an additional intervenient in brain injury by UCB. Next we decided to further clarify

how this increased production of glutamate and NO could be involved in UCB-induced

nerve cell injury. To answer this question we used primary cultures of cortical neurons in

order to better elucidate the molecular mechanisms involved.

3.2. NMDA receptors and NO are implicated in UCB-induced neurite impairment and in cell death Cortical neurons cultured for 3 DIV were exposed, during 24 h, to either no

addition (control), or 50 µM UCB, in the absence or presence of 1 µM MK-801 (a NMDA

glutamate-subtype receptor antagonist) or 100 µM L-NAME (a non-specific NO synthase

inhibitor). Following UCB exposure, we started by evaluating neuronal demise by using

PI followed by the determination of neurite extension and ramification as we wanted to


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assess if glutamate and NO were responsible partners in neuritic arborisation impairment

by UCB.

As shown in Figure IV.3, either MK-801 or L-NAME added at the same time as

UCB were able to totally prevent (p<0.01) the necrotic-like cell death that showed to

duplicate by UCB over control values in immature neurons.

Fig. IV.3. UCB-induced neuronal death is fully abrogated by MK-801 and L-NAME. Cortical neurons cultured for 3 days in vitro (DIV) were exposed for 24 h, to either no addition (control) or 50 µM UCB, in the absence or presence of 1 µM MK-801 or 100 µM L-NAME, and further incubated with 75 μM PI as described in Methods. (A) The percentage of PI-positive cells was calculated and expressed as mean ± SEM from three independent experiments performed in triplicate. (B) Representative results of one experiment are shown. Scale bar represents 40 µm. **p<0.01 vs. respective control; §§p<0.01 vs. UCB alone.

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Moreover, the same was observed by either MK-801 or L-NAME in protecting the

UCB-induced reduction in neurite extension and ramification provoked by UCB in both 4

DIV (MK-801, p<0.01 and p<0.05, respectively; L-NAME, p<0.01 for both) and 18 DIV

neurons (MK-801, p<0.05 for both; L-NAME, p<0.05 and p<0.01, respectively), as shown

in Figure IV.4. These results provide proof of concept that glutamate and NO participate

in UCB-induced neurodevelopment abnormalities implicated in UCB-enduring harmful

consequences and that microglia, as major sources of these neurotoxic molecules upon

UCB stimulation, participate in its harmful effects. Based on the antioxidant properties of

GUDCA in neurons (Brito et al., 2008a; Vaz et al., 2010) and on the immunosuppressive

properties of both GUDCA and IL-10 (Fernandes et al., 2007) we decided to investigate

their potentialities in saving neuritic arborisation from UCB harmful effects.

Fig. IV.4. UCB-elicited impairment in neuritic outgrowth at 4 and 18 days in vitro (DIV) is mediated by NO and overstimulated NMDA receptors. Cortical neurons cultured for 3 DIV were exposed for 24 h, to either no addition (control) or 50 µM UCB, in the absence or presence of 1 µM MK-801 or 100 µM L-NAME as described in Methods. Incubation medium was removed and neurons were allowed to culture until 18 DIV. Neurite extension (A) and number of nodes (B) were identified by immunolabeling for MAP-2, quantified by ImageJ and expressed as arbitrary units (mean ± SEM). Representative results of one experiment regarding 4 DIV neurons is shown in C. Scale bar represents 40 µm. *p<0.05 and **p<0.01 vs. respective control; §p<0.05 and §§p<0.01 vs. UCB alone.


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3.3. UCB-elicited accumulation of extracellular glutamate is reduced by both GUDCA and IL-10, but not abolished

Taking into account the important role of glutamate NMDA receptors in UCB

neurotoxicity portrayed by the above data, we then evaluated the effects of GUDCA and

IL-10 upon UCB-induced glutamate release by cultured neurons. As shown in Figure

IV.5, GUDCA significantly reduced (~30%, p<0.05) the efflux of glutamate by UCB-

treated immature neurons, an effect even more marked (~45%, p<0.01) when we used

IL-10. Nevertheless, although these compounds significantly drop the extracellular

accumulation of glutamate they were unable to prevent it.

Fig. IV.5. UCB-induced extracellular accumulation of glutamate is prevented by both GUDCA and IL-10. Cortical neurons cultured for 3 days in vitro (DIV) were exposed for 24 h, to either no addition (control) or 50 µM UCB, in the absence or presence of 50 µM GUDCA or 10 ng/mL IL-10. Results are mean ± SEM from three independent experiments performed in triplicate. **p<0.01 vs. respective control; §p<0.05 and §§p<0.01 vs. UCB alone.

3.4. GUDCA or IL-10 pre-treatment counteracts impairment of neurite outgrowth and ramification, as well as cell death in UCB-treated neurons

Considering the effects of UCB in neurons, above described, and the protective

effects displayed by GUDCA and IL-10 in these cells we next investigated whether these

compounds reveal ability to abrogate UCB-induced necrotic-like cell death in immature

neurons, as well as neuritic changes. Both molecules besides abolishing cell-death

(p<0.01, Figure IV.6) showed efficacy in preventing short-term deficits in neurite

extension (GUDCA, p<0.05; IL-10, p<0.01) as depicted in Figure IV.7.

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Fig. IV.6. GUDCA or IL-10 pre-treatment counteracts cell death in UCB-treated immature neurons. Cortical neurons cultured for 3 days in vitro (DIV) were exposed for 24 h, to either no addition (control) or 50 µM UCB, in the absence or presence of 50 µM GUDCA or 10 ng/mL IL-10, and further incubated with 75 μM PI as described in Methods. (A) The percentage of PI-positive cells was calculated and expressed as mean ± SEM from three independent experiments performed in triplicate. (B) Representative results of one experiment are shown. Scale bar represents 40 µm. **p<0.01 vs. respective control; §§p<0.01 vs. UCB alone.

The same was verified in the maintenance of neurite ramification, where

significant protection was manifested by both GUDCA (p<0.05) and IL-10 (p<0.01) in 4

DIV neurons. Interestingly, even after removing the UCB stimulus and additionally

culturing neurons until 18 DIV, the protective effects of GUDCA and IL-10 on UCB-

induced long-term neuritic outgrowth impairment were still noticeable (p<0.05).


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Fig. IV.7. GUDCA or IL-10 pre-treatment counteracts impairment of neurite outgrowth at 4 and 18 days in vitro (DIV) in UCB-treated immature neurons. Cortical neurons cultured for 3 DIV were exposed for 24 h, to either no addition (control) or 50 µM UCB, in the absence or presence of 50 µM GUDCA or 10 ng/mL IL-10 as described in Methods. Incubation medium was removed and neurons were allowed to culture until 18 DIV. Neurite extension (A) and number of nodes (B) were identified by immunolabeling for MAP-2, quantified by ImageJ and expressed as arbitrary units ± SEM. Representative results of one experiment regarding 4 DIV neurons is shown in C. Scale bar represents 40 µm. *p<0.05 and **p<0.01 vs. control; §p<0.05 and §§p<0.01 vs. UCB alone.

3.5. UCB decreases the expression of pre-synaptic proteins and this event is abrogated by GUDCA, but not by IL-10

Previous results have proven that UCB can adversely affect spine formation and

synapse establishment (Fernandes et al., 2009). Moreover, UCB-induced impairment of

synaptic plasticity has already been acknowledged (Chang et al., 2009). Therefore, we

decided to examine if UCB can modify the expression of proteins such as synaptophysin

and SNAP-25, which are involved in synaptogenesis and synaptic vesicle assembly. Our

results showed that UCB led to a significant reduction in both protein levels (0.9 fold,

p<0.05 and 0.8 fold, p<0.01, respectively). In line with the aforementioned benefits,

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GUDCA prevented the UCB synaptotoxicity from occurring (p<0.05), while a slight and

non-significant effect was produced by IL-10 pretreatment (Figure IV.8).

Fig. IV.8. UCB down-regulation of synaptophysin and SNAP-25 protein expression in immature neurons is prevented by GUDCA. Cortical neurons cultured for 3 days in vitro (DIV) were exposed for 24 h, to either no addition (control) or 50 µM UCB, in the absence or presence of 50 µM GUDCA or 10 ng/mL IL-10. Crude synaptosomal fractions were analyzed by western blotting with antibodies specific for synaptophysin and SNAP-25. The intensity of the bands was quantified by scanning densitometry, standardized with respect to β-actin protein expression. Graph bars represent the fold change values (mean ± SEM) from three independent experiments performed in duplicate for synaptophysin (A) and SNAP-25 (B). Representative results of one experiment regarding synaptophysin and another concerning SNAP-25 are shown in (C). **p<0.01 vs. respective control; §p<0.05 vs. UCB alone.

Interestingly, GUDCA even revealed to up-regulate synaptophysin and SNAP-25

expression. Thus, GUDCA evidence to be a more promising therapeutical approach in

hyperbilirubinemia than IL-10 due to its broader benefits and induced increase in pre-

synaptic proteins that regulate synaptic vesicle docking, as well as membrane fusion and

fission (Gray et al., 2010).


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3.6. Hampering of UCB-induced NO and glutamate production, as well as cell death by GUDCA was reproduced in hippocampal slices

Given the promising protective effects of GUDCA regarding UCB neurotoxicity,

verified for purified cultures, we became interest in returning to the singular physiological

conditions provided by the hippocampal slice model to verify if the observed effects were

reproduced in this more complex model. We were particularly interested in studying the

pathways previously shown as activated in microglial cells upon UCB exposure, namely

glutamate and NO production. Therefore, we assessed extracellular accumulation of

glutamate and NO release, as described in Figure IV.9, and found that GUDCA was able

to counteract their production (p<0.01).

Fig. IV.9. GUDCA hampers UCB-induced NO and glutamate production in organotypic-cultured hippocampal slices. Hippocampal slices cultured for 3 days in vitro were exposed for 24 h, to either no addition (control) or 50 µM UCB, in the absence or presence of 50 µM GUDCA. (A) Tissue concentrations of glutamate were evaluated using a colorimetric assay and expressed as mean ± SEM fold change compared with control conditions. (B) NO production was estimated by the quantification of nitrite levels and expressed as mean ± SEM from three independent experiments performed in duplicate. *p<0.05 and **p<0.01 vs. control; §§p<0.01 vs. UCB alone.

Moreover, similar neuroprotective effects were demonstrated by the abrogation of

PI uptake (p<0.01) in GUDCA-treated slices prior to UCB incubation (Figure IV.10),

further highlighting the therapeutic potential of this particular bile acid.

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Fig. IV.10. GUDCA hampers UCB-induced cell death in organotypic-cultured hippocampal slices. Hippocampal slices cultured for 3 days in vitro were exposed for 24 h, to either no addition (control), or 50 µM UCB, in the absence or presence of 50 µM GUDCA, and further incubated with 2 μg/mL PI as described in Methods. (A) Representative results of one experiment are shown. Scale bar represents 40 µm. (B) Graph bars represent the fold change values (mean ± SEM) from three independent experiments performed in duplicate. **p<0.01 vs. respective control; §§p<0.01 vs. UCB alone.

4. Discussion In this study we demonstrate, for the first time, that microglia modulate UCB-

induced neurotoxicity in organotypic-cultured hippocampal slices. This modulation

involves glutamate and NO as key mediators in the injurious effects prompted by UCB.

Furthermore, our study depicts the neuroprotective properties of GUDCA and IL-10,

revealing that their potential to prevent neuronal damage observed in pure neuronal

cultures is reproduced in the more complex system of organotypic cultures. In addition,

their ability to abrogate the neurodevelopmental abnormalities caused by neonatal


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hyperbilirubinemia reinforces their possible use as new therapeutic approaches in this,

still, life-threatening condition.

Previous reports have shown that UCB interacts with whole nerve cell and

mitochondrial membranes disrupting its redox status (Rodrigues et al., 2002b; Rodrigues

et al., 2002c) and that several oxidative biomarkers are elicited in neuronal (Vaz et al.,

2010) and astroglial cells (Brito et al., 2008b), as well as in synaptosomal membrane

systems (Brito et al., 2004). Moreover, unpublished results from our group support the

increase in NO production upon UCB exposure in neurons (personal communication Ana

Rita Vaz, 2010) and microglia, where the production is further amplified by UCB-treated

neuronal conditioned medium (unpublished results). Indeed, NO appears to be involved

in numerous physiological and pathological processes (Bishop and Anderson, 2005).

Several authors have evidenced that activated microglia releases NO following induction

of nitric oxide synthase (Kawase et al., 1996) and that this radical mediates neural injury

(Golde et al., 2002). On the other hand, the generation of an oxidative burst by microglia

in response to injury may also constitute a measure to promote the clearance of

damaged cells from the CNS (Kawase et al., 1996). The results displayed in this study

support the role of glutamate and NO as important neurotoxic molecules. Moreover, as

was shown in the ex vivo organotypic-cultured hippocampal slice model, their production

upon UCB exposure may be attributed to microglial cells. Accordingly, blocking the

action of both molecules abolished the toxic effects of UCB in our primary neuron culture

model.

However, the role of microglia in glutamate homeostasis upon UCB exposure

seems to be more complex, as opposing effects were observed when evaluating

glutamate content in tissue homogenates or incubation media from organotypic-cultured

hippocampal slices. In fact, results from tissue samples point to microglia as the most

important glutamate source, nevertheless, extracellular accumulation of glutamate in

incubation media from microglia-depleted slices is enhanced, suggesting that microglia

are actually promoting glutamate uptake. Indeed, activated microglia have been shown

to up-regulate glutamate transporter-1 (GLT-1) in response to motoneuron injury in

axotomized rat facial nucleus (Lopez-Redondo et al., 2000) and also as a defensive

mechanism against herpes simplex virus infection (Persson et al., 2007). Additionally,

inflammatory products such as tumor necrosis factor-α may similarly induce GLT-1

expression in microglia (Persson et al., 2005) while glutamine synthetase, the enzyme

responsible for converting glutamate into the less toxic glutamine, is also expressed in

activated microglia (Chretien et al., 2002; Rimaniol et al., 2000). This inducible profile of

microglia contrasts with the constitutive expression of glutamate transporters in

astrocytes and can be regarded as a compensatory mechanism to limit the deleterious

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consequences of microglial activation in the brain (Gras et al., 2006). Our hypothesis is

that microglia initially exerts a neuroprotective action towards UCB by promoting

glutamate clearance, either by themselves or by engaging astrocytes in this function, as

previously observed (Tilleux et al., 2009). However, despite microglial efforts to limit

glutamate concentration, the ultimate outcome is an increase in its extracellular levels,

possibly due to the failure of the clearance mechanisms or to the increased production

by either glial or neuronal cells upon prolonged exposure times. A growing body of

evidence underscores glutamate’s key role in microglial neurotoxicity (Barger and Basile,

2001; Liang et al., 2008). Furthermore, several reports link oxidative stress and

glutamate-mediated excitotoxicity (Bal-Price and Brown, 2001; Barger et al., 2007; Brito

et al., 2010; Golde et al., 2002), which is consistent with our results demonstrating that

glutamate and NO participate in UCB-induced neuronal demise. In fact, earlier studies

have already reported that UCB-induced neurotoxicity is mediated by glutamate

receptors (Grojean et al., 2000; Grojean et al., 2001; Hanko et al., 2006; Hoffman et al.,

1996).

Glutamate can cause alterations in dendritic outgrowth (Esquenazi et al., 2002;

Mattson, 2008), even in immature neurons (Monnerie et al., 2003), as well as NO, that

was shown to induce synapse loss (Sunico et al., 2010). Interestingly, our previous

studies evidenced impairment of neurite extension and ramification in immature cortical

neurons exposed to UCB that is sustained through cell maturation (Falcão et al., 2007).

Changes in dendritic and axonal arborisation were also observed in UCB-treated

immature hipppocampal neurons (Fernandes et al., 2009). Our novel results bridge

those two findings, demonstrating a causal relation between UCB-induced impairment of

neurite outgrowth, NMDA receptor overstimulation, and NO overproduction. This aspect

gains additional relevance if we consider that glutamate efflux is enhanced in neurons

and glial cells (Falcão et al., 2005; Fernandes et al., 2004) exposed to UCB, and its

uptake inhibited in neurons (Silva et al., 1999; Silva et al., 2002). This finding is

particularly important given the fact that neonatal hyperbilirubinemia has been

associated to the development of mental disorders (Dalman and Cullberg, 1999) like

schizophrenia (Hayashida et al., 2009; Miyaoka et al., 2000), and can impact on learning

and memory (Zhang et al., 2003). UCB can adversely affect synapse establishment

since it interferes with spine formation and reduces growth cone area (Fernandes et al.,

2009). Accordingly, UCB exposure leads to impairment of neurotransmitter release in

synaptic vesicle membranes (Roseth et al., 1998), modification of long-term synaptic

plasticity (Chang et al., 2009), and decreased synaptic activity in hippocampal slices

(Hansen, 1994). These studies are consistent with our results regarding decreased pre-

synaptic protein expression upon UCB exposure. Actually, glutamate’s implication in


127

neuritogenesis and synaptogenesis has already been acknowledged (Mattson et al.,

1988).

In this paper we report that UCB can lead to a diminished expression of the pre-

synaptic proteins synaptophysin and SNAP-25. Synaptophysin is a calcium binding

protein expressed on pre-synaptic vesicles and SNAP-25 is anchored in the synaptic

terminal plasma membrane. These proteins are essential for synaptic vesicle fusion to

the pre-synaptic plasma membrane, thus participating in synapse establishment and

neurotransmitter release (Wang and Tang, 2006). A decrease in synaptic protein

expression is regarded as a biomarker of altered neuronal development. In fact,

synaptophysin expression is altered in epilepsy (Xu et al., 2009) and changes in proteins

relevant to synaptic transmission and axonal transport have been coupled to

neuroinflammation in Parkinson's disease (PD) (Chung et al., 2009). Cytoskeleton

abnormal assembly, loss of dendrites and axons and impairment of neurotransmission

can cause disruption of synaptic connectivity and instigate neurodegenerative diseases

like Alzheimer’s disease (Evans et al., 2008) or PD (Benitez-King et al., 2004), and

psychiatric illnesses such as schizophrenia (Brennaman and Maness, 2008).

GUDCA is the major product of ursodeoxycholic acid (UDCA) catabolism

(Rudolph et al., 2002). UDCA and its conjugates have demonstrated neuroprotective

effects by the stabilization of mitochondrial membranes (Solá et al., 2002), inhibition of

mitochondrial swelling (Rodrigues et al., 2000b), cytochrome c release (Rodrigues et al.,

2000a) and, ultimately, by the decrease of apoptotic cell death (Silva et al., 2001). UDCA

oral administration induces a rapid and sustained decrease in plasma UCB

concentrations in Gunn rats, the animal model used to study bilirubin encephalopathy

(Cuperus et al., 2009). Recent reports have indicated potential benefits for GUDCA at

preventing UCB-induced protein oxidation, lipid peroxidation, impairment of glutathione

homeostasis and neuron cell death (Brito et al., 2008a), or mitochondrial respiratory

chain dysfunction by UCB and restoration of cellular antioxidant potential (Vaz et al.,

2010). Our group has also evidenced that both GUDCA and IL-10 can modulate

astrocytic reactivity to UCB decreasing the elicited inflammatory properties and reducing

cell death (Fernandes et al., 2007). The anti-inflammatory cytokine IL-10 has already

been demonstrated to reduce glial activation (Ledeboer et al., 2000) and to exert

neuroprotective effects (Bachis et al., 2001; Park et al., 2007). IL-10 is also able to down-

regulate microglial NO production, thus contributing to the recovery of neurite outgrowth

(Rozenfeld et al., 2003). Our results show that UCB-induced deleterious effects on

neuronal survival and neurite impairment are also prevented by GUDCA and IL-10.

Protective properties of these compounds on the cellular efflux of glutamate, elicited by

UCB, emphasize the protective effects observed since we have here demonstrated the

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essential role of glutamate and NO in UCB-evoked alterations in network dynamics. To

the best of our knowledge, only one other molecule, taurine, has evidenced protective

effects against UCB-mediated neuronal damage (Zhang et al., 2010). It is also very

important to acknowledge that neuroprotection by GUDCA in the purified neuronal

culture, was kept in organotypic-cultured hippocampal slices, suggesting its usefulness

in vivo.

Given the fact that both GUDCA and IL-10 showed to prevent UCB-induced

extracellular accumulation of glutamate, we expected that both molecules would exert

protective effects on pre-synaptic protein expression. Surprisingly, only GUDCA proved

to be effective at this level, which led us to the hypothesis that GUDCA might be acting

on different mechanisms beyond glutamate-mediated neuronal damage. One possible

explanation might be that GUDCA has recognized anti-oxidant properties (Brito et al.,

2008a; Vaz et al., 2010) and, in fact, oxidative stress is associated with synaptogenesis

and neuritogenesis alterations (Sunico et al., 2010), while IL-10 is more notorious for its

anti-inflammatory capacities (Ledeboer et al., 2000). Moreover, GUDCA and IL-10 have

also shown to modulate astroglial reactivity to UCB following different mechanisms

(Fernandes et al., 2007).

In conclusion, the results obtained in this study portray an interesting and

complex role for microglia in glutamate homeostasis upon UCB exposure. These cells

induce glutamate clearance as a first attempt to limit the excitotoxic neuronal damage

but, on the other hand, also contribute for the pool of released glutamate culminating in

an extracellular accumulation of this molecule. Moreover, our study evidences microglial

cells as a source of NO upon UCB exposure. Our work is the first to establish the

involvement of glutamate and NO in UCB-induced impairment of neurite extension and

ramification, thus substantiating target-driven approaches which may reveal important in

the prevention of long-term neurodevelopment disabilities. Most important is the

neuroprotective effects of GUDCA and IL-10 in hyperbilirubinemia as they are able to

prevent the UCB-induced increase in glutamate secretion and, through this or other

mechanisms, counteract the deleterious effects of UCB on neuronal network dynamics

and cell death. GUDCA has an additional benefit in preventing down-regulation of pre-

synaptic protein expression, an event that may underlie UCB-evoked alterations in

synaptic plasticity.

In conclusion, our data reveal microglia, glutamate and NO as potential targets

for more directed and effective early-therapeutic interventions, and GUDCA as a

promising medicine in the management of neonatal hyperbilirubinemia.


129

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Chapter V

FINAL CONSIDERATIONS

Final Considerations

137

1. Concluding remarks and perspectives The general goal of this thesis was to explore if and how migroglial cells

participate in the neurodegeneration due to UCB encephalopathy. In fact, previous

studies had already demonstrated that UCB promoted a marked inflammatory response

in astrocytes with the release of pro-inflammatory cytokines and glutamate, and that this

inflammatory reaction was prompted by the activation of several signalling pathways

(Fernandes et al., 2007a; Fernandes et al., 2006; Fernandes et al., 2004). Moreover, this

immunostimulant effect was proven to be more relevant in astrocytes than in neurons

exposed to UCB, while for this last cell type cell death by both necrosis and apoptosis

was the most likely expected outcome (Falcão et al., 2005; Falcão et al., 2006).

Nevertheless, and despite the notorious contribution of microglia to several neonatal

pathological conditions such as hypoxic- ischemic injury (McRae et al., 1995; Vexler and

Yenari, 2009), little was known about the involvement of microglia in UCB-elicited

toxicity. Early reports had demonstrated that UCB may alter the function of various cells

of the immune system (both in vivo and in vitro) (Vetvicka et al., 1985; Vetvicka et al.,

1991). In addition, an increase in phagocytosis of both peripheral blood granulocytes and

monocytes after UCB treatment was reported by Miler et al. (1985). Our group was the

first to demonstrate that UCB also causes microglial activation characterized by

morphological alterations, increased secretion of pro-inflammatory cytokines and

glutamate as well as microglial cell death, after a short exposure period to UCB (Gordo

et al., 2006). Indeed, when compared to other brain cells, microglia revealed to be the

most responsive ones to UCB insult (Brites et al., 2009). However, a more detailed

characterization of microglial reactivity profile to UCB was needed to further elucidate the

molecular and cellular mechanisms of UCB-induced toxicity.

Using an experimental model of isolated primary cultures of microglia we

described the temporal profile of microglial reactivity towards UCB. We observed, for the

first time, that UCB induces an increase in the phagocytic properties of microglia,

followed by a shift into a rather inflammatory response with prolonged exposure time. So,

we next characterized the inflammatory reaction elicited by UCB in those cells and found

that MAPKs phosphorylation is an essential step for NF-κB nuclear translocation and that

these events occur upstream of the release of inflammatory mediators such as TNF-α,

IL-1β and IL-6, thus probably signalling this release. Moreover, additional inflammatory

indicators were displayed by UCB-reactive microglial cells such as COX-2 up-regulation

and MMP-2 and -9 increased activation. Furthermore, we provided evidence that UCB-

induced cytokine secretion, particularly IL-1β, may participate in MMPs activation

suggesting that these events might be reciprocally regulated.

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Interestingly, the alternation of the different activation stages of microglia along

time in the presence of UCB, phagocytic and inflammatory, seem to indicate that

microglia reacts towards UCB insult firstly with a phagocytic response, in an attempt to

constrain the lesion extent and comprising a neuroprotective measure. Upon prolonged

UCB exposure periods, either a shift on global microglia reaction occurs or two distinct

sub-populations of microglial cells may co-exist, one directed at eliminating the damaged

cells by phagocytosis, and another that engaged a more delayed inflammatory response.

The fact that cell death by both apoptosis and necrosis arouses after the observed

phagocytic response in microglia further corroborates the stated hypothesis. Accordingly,

clearance of tissue debris performed by microglia following injury has been

demonstrated in several disease models and might constitute a regenerative measure

(Napoli and Neumann, 2009; Neumann et al., 2009). Our findings add on to the exciting

concept that microglia displays functional plasticity (Graeber and Streit, 2009; Schwartz

et al., 2006) and emphasize the role of these cells as active sensors of the brain that

adapt their reactive phenotypes to the environmental circumstances (Hanisch and

Kettenmann, 2007).

Nevertheless, the brain is not compartmented and cellular interactions are

essential regulators of many critical functions in the healthy and the diseased brain

(Biber et al., 2007). Taking this into consideration, we found valuable to investigate how

the interplay between microglia and other nerve cells could modulate the effects induced

by UCB. In order to achieve this goal we used two different experimental models:

conditioned media, to evaluate the effect of soluble factors, and mixed neuron-glia

cultures, to assess proximity-dependent interactions. Our results showed that soluble

factors released by astrocytes exposed to UCB dampen microglial production of

inflammatory cytokines such as TNF-α and IL-1β, diminish MMP-9 activation and prevent

cell death. Therefore, microglia, when isolated, respond intensively to UCB stimulation,

but previously activated astrocytes seem to drive microglia to refrain its response. Other

authors have postulated that this is a feasible mechanism to prevent excessive brain

inflammation (Min et al., 2006). Interestingly, UCB-treated neuron conditioned medium

produced a similar down-regulation on the production of inflammatory mediators by

microglia but enhanced NO generation, MMP-9 activation and led to cell demise. In

addition, we demonstrated that microglia’s phagocytic abilities were further enhanced

when these cells were exposed to conditioned medium derived from UCB-exposed

neurons, instead of direct UCB stimulation. Such finding suggests that UCB-injured

neurons might be signalling microglia to engage a phagocytic phenotype in an attempt to

constrain lesion extent but also enhanced its inflammatory potential ultimately leading to


139

microglia demise. This hypothesis was further strengthened by the results regarding

UCB-deleterious effects on neurite network and neuronal cell death when neurons were

cultivated in close proximity with microglial cells. In fact, these studies showed that the

previously observed UCB-induced neurite outgrowth impairment and cell demise (Falcão

et al., 2007) were abrogated in the presence of microglia. This apparent protection could

be the result of the phagocytic clearance of UCB-damaged neurons, similarly to the

events described in other disease models as revealed by the phagocytosis of cellular

debris or amyloid deposits by microglia in MS (Takahashi et al., 2005) and AD (Simard et

al., 2006), respectively.

The results obtained pertaining neuron-glia dynamics upon UCB exposure

prompted us to explore the role of microglia in UCB-induced neurotoxicity in a more

complex model, the organotypic slice cultures. These studies were performed in the

hippocampus, one of the brain areas affected in kernicterus (Shapiro, 2003; Shapiro,

2005) that showed increased vulnerability to UCB’s toxic effects as proven by the

impairment of long-term synaptic plasticity found by Chang et al. (2009) in this brain

area. By the selective depletion of microglia from organotypic-cultured hippocampal

slices we were able to determine that these cells are the major sources of NO upon UCB

stimulation and that they significantly interfere with glutamate homeostasis. The inducible

expression of glutamate transporters (Lopez-Redondo et al., 2000; Persson et al., 2005)

as well as the presence of glutamine synthetase in microglia (Rimaniol et al., 2000) has

been previously reported. Our results suggest an initial neuroprotective role for microglia

as glutamate scavengers. However, despite microglial efforts to limit glutamate

concentration, the ultimate outcome is an increase in its extracellular levels and tissue

accumulation, possibly due to the failure of the clearance mechanisms or to increased

production by either glial or neuronal cells upon prolonged exposure times to UCB.

Interestingly, glutamate had already been stressed out as an important mediator

of UCB-induced neurotoxicity (Johnston, 2005) since its astrocytic uptake is inhibited in

astrocytes (Silva et al., 1999) prolonging its presence in the synaptic cleft and

engendering NMDA overactivation and consequent excitotoxic death (Grojean et al.,

2000; Grojean et al., 2001; McDonald et al., 1998). Moreover, oxidative stress has also

been demonstrated to be involved in UCB-induced injury (Brito et al., 2004; Brito et al.,

2008; Vaz et al., 2010). More recently the two events were shown to be associated

during UCB cytotoxicity since overactivation of glutamate receptors seems to mediate

oxidative damage in neurons (Brito et al., 2010). In addition, oxidative stress was also

shown to mediate UCB-induced degeneration of excitatory synaptic terminals in the

auditory brainstem (Haustein et al., 2010). Therefore, we decided to explore the role of

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glutamate and NO in the impairment of neurite extension and ramification and in

neuronal death in order to elucidate the molecular mechanisms triggering

neurodevelopmental changes. Our studies clearly demonstrate the key role of NO and

overactivation of NMDA receptors in UCB-induced impairment of neuritic outgrowth and

neuronal demise, providing exciting new targets for the management of neonatal

hyperbilirubinemia. Our results also indicated that UCB decreased the expression of pre-

synaptic proteins, which are essential for synapse establishment and neurotransmitter

release, and are consistent with the findings showing that UCB can also adversely affect

synapse establishment (Fernandes et al., 2009), and cause alterations of synaptic

plasticity (Chang et al., 2009). Abnormal cytoskeleton assembly, loss of dendrites and

axons and impairment of neurotransmission can cause disruption of synaptic

connectivity and instigate neurodegenerative diseases and mental disorders like

schizophrenia (Benitez-King et al., 2004; Brennaman and Maness, 2008). Thus,

association between neonatal hyperbilirubinemia and proneness to mental disorders in

later life seems to be a reasonable possibility.

In this study we also evaluated the therapeutic potential of GUDCA and IL-10 and

found that these molecules were able to counteract the above mentioned deleterious

effects prompted by UCB, namely network dynamics impairment and neuronal cell

death, probably by the reduction of glutamate extracellular accumulation. In addition,

GUDCA proved to be more effective than IL-10 in preventing the down-regulation of pre-

synaptic proteins’ expression. Besides, GUDCA proved to also abrogate the injurious

effect elicited by UCB not only in isolated neurons but also in the hippocampal slice

culture model, thus reinforcing its potential as a therapeutic approach in the

management of neonatal hyperbilirubinemia.

Collectively, the data presented in this thesis (Fig. V.1) provide interesting

findings regarding the implication of microglia in UCB-induced phenomena. Indeed,

different reactive phenotypes are engaged by these cells depending on the duration and

intensity of the toxic stimulus and on the modulation exerted by neighbouring cells,

reinforcing their remarkable functional plasticity.


141

Fig.V.1. Schematic representation of microglia reactivity to unconjugated bilirubin. Isolated microglial cells present a dual phenotype, phagocytic and inflammatory, that switch along unconjugated bilirubin (UCB) exposure, with the release of several inflammatory mediators and activation of diverse upstream signalling pathways (A). In addition, astrocytes exposed to UCB dampen microglia activation while neurons signal a phagocytic phenotype in microglia and enhance its inflammatory potential leading to cell demise (B). Moreover, in UCB-stimulated neuron-microglia mixed cultures the neurotoxic effects of UCB are prevented, suggesting that stressed neurons promote a neuroprotective clearance function in microglia (C). Finally, microglia revealed to participate in glutamate homeostasis and to induce the release of nitric oxide (NO) in UCB-treated hippocampal slices (D), important molecules in UCB-induced neurotoxicity. Moreover, our results identify some of the therapeutic actions of interleukin (IL)-10 and glycoursodeoxycholic acid (GUDCA).

Mixed culturesConditioned media

UCB

IL‐1βIL‐6MMP‐9LDH leakage

IL‐1βand TNF‐αIL‐6MMP‐9LDH leakagePhagocytosis

UCB

Neurite outgrowthimpairment

Cell death

15 min0 h 30 min 2 h 4 h 8 h 12 h 24 hUCB

MAPKs NF‐κΒ Phagocytosis

Cell death

Inflammation

COX‐2

IL‐1β

TNF‐α

MMP‐2

MMP‐9

Isolatedmicroglia

IL‐6

Organotypic‐culturedhippocampal slices

UCB

Glu

UCB

Neurite outgrowthimpairment

Cell death

GUDCA

NO

Glu

SNAP‐25Synaptophysin

IL‐10

Isolatedneurons

A

B C

D

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However, some questions are left unanswered and remaining to be clarified.

Taking advantage of the singular physiological conditions provided by the hippocampal

slice model, it would be interesting to evaluate exactly which glial cells participate more

actively in the neurodegeneration process elicited by UCB. In addition, it would also be

relevant to study the ability of UCB to promote cell proliferation and the identification of

the most affected ones. In fact, one could speculate that microglia would top the list

given its characteristic capacity to proliferate and migrate towards the injury site upon

brain injury (Heppner et al., 1998; Ladeby et al., 2005). Indeed, the question whether

microglia can migrate towards UCB in the icteric brain should also be addressed using

this model. On the other hand, in vitro models for evaluation of microglial chemotactic

properties could also reveal useful to estimate if UCB exposure can alter microglial ability

to migrate towards known toxic chemoattractants. Finally, given the extraordinary

therapeutic potential revealed by GUDCA and its previously observed ability to prevent

astrocytic immunostimulation by UCB (Fernandes et al., 2007b), it would also be

pertinent to further investigate the implication of microglia in the protective effects elicited

by this molecule in slice cultures, performing additional studies in microglia-depleted

slices. As in other brain diseases, the main question left unanswered regarding microglial

reactivity in hyperbilirubinemia is to what extent silencing microglial activation might be

beneficial or detrimental. Indeed, a growing body of evidence supports the notion that

microglial cellular activities and inflammatory responses are mostly beneficial, becoming

potentially destructive when the strict regulatory control upon microglia is disrupted,

either by pathologic events or by dystrophy (Harry and Kraft, 2008).


143

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