UNIVERSITI PUTRA MALAYSIA

Morphological and Proliferative Responses of Rat Microglia Cells to Lipopolysaccharide and β-Amyloid

ALIREZA BADIEI

FPV 2008 9

Morphological and Proliferative Responses of Rat Microglia Cells to Lipopolysaccharide and β-

Amyloid

ALIREZA BADIEI

Master of Science Universiti Putra Malaysia

November 2008

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science

MORPHOLOGICAL AND PROLIFERATIVE RESPONSES OF RAT MICROGLIAL CELLS TO LIPOPOLYSACCHARIDE AND BETA AMYLOID

By

ALIREZA BADIEI

June 2008

Chairman : Sharmili Vidyadaran, PhD

Faculty : Faculty of Medicine and Health Sciences

Microglia are the residential macrophages of the central nervous system

(CNS) and are sensitive to any changes in their environment. There is rapid

microglial activation in most pathological conditions in CNS. The association

of activated microglia with various inflammatory and neurodegenerative

diseases are well documented. In this study we evaluated the in vitro

responses of microglia to lipopolysaccharide (LPS) or beta amyloid (Aβ) in an

attempt to further define the activation of microglia. LPS is a potent activator

of monocytes and macrophages and Aβ is used to activate microglia in vitro

into a neurotoxic phenotype distinguished by secretion of proinflammatory

molecules. Microglia were cultured from Sprague-Dawley neonatal rats and

purity of culture determined using a common marker for microglia, lectin.

Morphological changes following LPS or Aβ treatment were evaluated using

fluorescent and confocal microscope. Treated microglia assumed a

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deramified shape with a condensed cytoplasm, typical of activated microglia.

Stimulation of microglia with LPS or Aβ also resulted in significant

ultrastructural changes. Transmission electron microscopy revealed an

increased number of enlarged, elongated and swollen mitochondria. There

were also some changes in the appearance of the endoplasmic reticulum.

Untreated microglia displayed mainly smooth endoplasmic reticulum (SER)

whereas LPS-treated cells displayed polyribosomes and rough endoplasmic

reticulum (RER). It is therefore assumed that LPS-treated microglia have an

increased ability in synthesising protein, some of which may be secretory

molecules necessary for inflammation. Aβ-treated microglia also displayed

more RER compared to control, but lesser compared to LPS. The nucleus in

treated microglia appeared enlarged in comparison to untreated cells. The

number of cells following treatment revealed that microglia are more viable

following LPS treatment compared to Aβ. Immunophenotyping assays

demonstrated upregulation of MHC II and CD40 by microglia following

treatment. Both MHC II and CD40 are implicated in antigen-presenting and

this result indicates that in comparison to LPS, Aβ may have more ability to

induce antigen-presenting capabilities in microglia. As shown by viability

counts, carboxy fluorescein succinimidyl ester (CFSE) staining also revealed

no microglia proliferation following treatment with LPS and Aβ. In conclusion,

our study illustrates activation-induced alterations in the morphology,

ultrastructure and cell surface phenotype of microglia, which were not

accompanied by proliferation of microglia. These changes represent the

range of effects that occur in microglia following activation. In an attempt to

culture microglia from adult rats for the purpose of determining the effects of

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aging on microglia responses, modifications were made on the established

protocol for neonatal microglia cultures. This includes growing adult cells on

poly-L-lysine-coated tissue culture flasks and substituting insulin in cell

culture media with insulin-transferrin-selenium (ITS). We were able to

successfully support the in vitro growth of adult microglia, which was also

enhanced with the addition of the growth factor M-CSF.

Key words: microglia, lipopolysacharide, beta amyloid, morphology,

proliferation, CD40/MHC II

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

Respon morfologi dan pembahagian pada mikroglial sel tikus kepada lipopolisakarida dan amyloid beta

Oleh

ALIREZA BADIEI

Jun 2008

Pengerusi : Sharmili Vidyadaran, PhD

Fakulti : Perubatan dan Sains Kesihatan

Mikroglia merupakan makrofaj residen dalam sistem saraf pusat. Mikroglia

dirangsang dengan cepat dalam kebanyakan keadaan patologi. Banyak

penulisan saintifik yang membuktikan hubungan di antara mikroglia dengan

inflamasi dan penyakit neurodegenerasi. Dalam kajian ini, kami menilai

gerakbalas mikiroglia ke atas rawatan dengan lipopolysaccharide (LPS) dan

beta amyloid (Aβ) secara in vitro untuk mengenalpasti cara pengaktifan

mikroglia dengan lebih lanjut lagi. Mikroglia dikultur daripada tikus neonat

Sprague- Dawley dan tahap ketulenan kultur ditentukan melalui lektin yang

merupakan satu molekul penunjuk umum pada sel mikroglial. Perubahan

morfologi mikroglia selepas rawatan dengan LPS dan Aβ ditentukan dengan

menggunakan mikroskop fluoresen dan konfokal. Mikroglia yang dirawat

dikenal sebagai sel yang kurang bercabang (deramified) dengan sitoplasma

yang kondens. Ini adalah keadaan lazim bagi sel mikroglia yang telah

menjadi aktif. Rangsangan dengan LPS dan Aβ juga menyebabkan

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perubahan ultra-struktur dan rupabentuk yang ketara. Mikroskop elektron

transmisi menunjukkan penambahan bilangan mitokondria yang

membengkak, memanjang dan membesar. Terdapat juga perubahan

rupabentuk pada reticulum endoplasma. Sel mikroglia yang tidak menerima

sebarang rawatan mempunyai kebanyakannya retikulum endoplama yang

licin (smooth endoplasmic reticulum, SER) dan apabila dirawat dengan LPS,

sel-sel mempamerkan poliribisom (polyribosome) dan reticulum endoplasma

kasar (rough reticulum endoplasmic, RER). Sel yang dirawat dengan Aβ juga

mempamerkan lebih banyak reticulum endoplasma kasar berbanding dengan

sel kawalan. Akhirnya, sel yang dirawat juga mempamerkan pembesaran

nukleus berbanding dengan sel yang tidak dirawat. Asei

“immunophenotyping” menunjukkan penambahan ekpresi molekul MHC

kelas dua dan CD40 pada permukan sel mikroglial sejurus menerima

rawatan. Tiada proliferasi diperhatikan selepas rawatan sel dengan LPS dan

Aβ, seperti yang telah ditunjukan melalui pewarnaan CSFE. Ringkasnya,

kajian kami telah menunjukkan perubahan ultrastruktur dan rupabentuk serta

“fenotip” permukaan sel-sel mikroglial sejurus selepas sel-sel tersebut

dirangsang. Perubahan-perubahan ini bukanlah disebabkan proliferasi sel.

Semua perubahan ini mewakili pelbagai easan yang terjadi kepada sel

mikroglial selepas diaktifkan. Untuk pengkulturan sel mikroglia dewasa bagi

tujuan kajian ke atas kesan penuaan kepada mikroglia, beberapa modifikasi

telah dijalankan kepada protokol pengkulturan mikroglia daripada tikus muda.

Pada amnya, ini termasuk pengkulturan mikroglia dewasa di dalam kelalang

kultur tisu yang terlebih dahulu disaluti dengan poly-L-lysine dan

menggantikan insulin di dalam media kultur sel dengan insulin-transferrin-

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selenium (ITS). Kami berjaya mengkultur sel mikroglia dewasa, dan ini

dibaiki lagi dengan penambahan faktor pertumbuhan, M-CSF.

Kata kunci: mikroglia, lipopolisakarida, amyloid beta, morfologi, proliferasi,

CD40/MHC II

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ACKNOWLEDGEMENTS

I would first like to record my gratitude to my supervisor, Dr. Sharmili

Vidyadaran for her supervision, advice, and guidance from a very early stage

of this research as well as giving me extraordinary experiences throughout

the work. I am indebted to her more than she knows.

Deepest gratitude is also due to the member of the supervisory committee,

Prof. Dr. Seow Heng Fong - without her knowledge and assistance this study

would not have been successful.

Many thanks go in particular to Dr. Rajesh Ramasamy, I am much indebted

for his help in the flow cytometry experiments and also valuable advice in

science discussion. I also gratefully thank Dr Maha Abdullah for her

guidance.

I gratefully acknowledge Professor Nemat Khansari for his advice, scientific

support. His involvement with his originality has triggered and nourished my

intellectual maturity that I will benefit from, for a long time to come.

Many thanks go in particular to Leong Pooi Pooi, Yip Wai Kien, Vahid

Sarmadi and Tong Chih Kong for their help in handling precise and delicate

equipment in the Immunology Lab. I also benefited by outstanding assistance

from my other lab mates: Peyman Amini, Jee Jap Meng, Mahdi Jaafarloo,

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Leslie, Homa Davoodi, Ngiow Shin Foong, Vincent Leong, Anthony and

Aishah.

I would also like to convey thanks to Universiti Putra Malaysia for providing

the financial support, graduate research fellowship (GRF) and laboratory

facilities.

My parents deserve special mention for their inseparable support and

prayers. My Father in the first place is the person who put the fundament my

learning character, showing me the joy of intellectual pursuit ever since I was

a child. My Mother is the one who sincerely raised me with her caring and

gently love. My brother and sisters and their families - thanks for being

supportive.

Finally, I would like to thank everybody who was important to the successful

realisation of thesis, as well as express my apology for not being able to

mention you personally.

I certify that an examination Committee has met on 26/11/2008 to conduct

the final examination of ALIREZA BADIEI on his Master of Science thesis

entitled “MORPHOLOGICAL AND PROLIFERATIVE RESPONSES OF RAT

MICROGLIAL CELLS TO LIPOPOLYSACCHARIDE AND BETA AMYLOID”

in accordance with Universiti Pertanian Malaysia (higher Degree) Regulation

1981. The Committee recommends that thestudent be awarded the Master of

Science.

Members of the examination Committee were as follows:

Elizabeth George, PhD Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairman) Fauziah Othman, PhD Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner) Ammu Kutty d/o G.K.,PhD Associated Professor International Medical University (External Examiner)

Goh Yong Meng, PhD Lecturer Faculty of Veterinary Medicine Universiti Putra Malaysia (Internal Examiner) HASANA MOHD. GHAZALI, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia

Date

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This thesis was submitted to the senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the supervisory Committee were as follows:

SHARMILI VIDYADARAN, PhD Lecturer Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairman)

SEOW HENG FONG, PhD Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Member)

HASANA MOHD GHAZALI, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia

Date: 12 February 2009

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DECLARATION

I declare that the thesis is my original work except for quotations and citations, which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institution.

________________ ALIREZA BADIEI

Date:

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TABLE OF CONTENTS

ABSTRACT ......................................................... Error! Bookmark not defined. ABSTRAK ........................................................... Error! Bookmark not defined. ACKNOWLEDGEMENTS ............................................................................ viii DECLARATION ............................................................................................. xii TABLE OF CONTENTS ............................................................................... xiii LIST OF TABLES ......................................................................................... xiii LIST OF FIGURES ...................................................................................... xvii LIST OF ABBREVIATIONS ......................................................................... xix CHAPTER 1 .................................................................................................... 1 Introduction .................................................................................................... 1

1.1. Background ...................................................................................... 1 1.2. Research questions .......................................................................... 6 1.3. Aims of this study ............................................................................. 6

CHAPTER 2 .................................................................................................... 7 Literature Review ........................................................................................... 7

2.1. Microglia ............................................................................................. 7 2.2. Origin of microglia ............................................................................... 7 2.3. Distribution of microglia in CNS ..................................................... 9 2.4. Resting and activated phenotype of microglia .................................... 10

2.4.1. Morphology .................................................................................. 11 2.4.2. Expression of surface receptors .................................................. 12 2.4.3. Proliferation .................................................................................. 15 2.4.4. Expression of proinflammatory mediators .................................... 16

2.5. Microglia activation: neuroprotective or neurotoxic? ........................... 18 2.5.1. Microglia are neurosupportive ...................................................... 19 2.5.2. Microglia and neurotoxicity .......................................................... 21 2.5.3. Microglia and neurodegenerative diseases ................................. 22

2.6. Senescence ....................................................................................... 24 2.7. Immunosenescence ........................................................................... 25

CHAPTER 3 .................................................................................................. 28 Materials & Methods .................................................................................... 28

3.1. Animals .............................................................................................. 28 3.2. Primary cell culture ................................................................................ 28

3.2.1. Media ........................................................................................... 28 3.2.2. Isolation of brain tissue ................................................................ 29 3.2.3. Mixed glia culture ......................................................................... 29 3.2.4. Cell harvesting ............................................................................. 30 3.2.5. Cell viability .................................................................................. 30 3.2.6. Lectin staining .............................................................................. 30

3.3. Treatment ........................................................................................... 31 3.3.1. Treatment with LPS ..................................................................... 31

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3.3.2. Treatment with Aβ ........................................................................ 31 3.4. Ultrastructural studies ......................................................................... 32

3.4.1. Treatment .................................................................................... 32 3.4.2. Sample preparation ..................................................................... 32

3.5. Immunophenotyping ........................................................................... 34 3.5.1. Sample preparation ..................................................................... 34 3.5.2. Acquisition and analysis by flow cytometry .................................. 34

3.6. CFSE proliferation assay .................................................................... 35 3.6.1. Sample preparation ..................................................................... 35 3.6.2. Acquisition and analysis by flow cytometry .................................. 35

3.7. Establishing adult microglia cultures .................................................. 36 3.7.1. Coating flasks with collagen and poly-L-lysine............................. 36 3.7.2. Replating non-adherent cells ....................................................... 37 3.7.3. Use of ITS in cell culture media ................................................... 37

3.8. Supplementing cell culture media with growth factors ........................ 37 3.8.1. GM-CSF ...................................................................................... 37 3.8.2. M-CSF ......................................................................................... 37

3.9. Statistical analysis .............................................................................. 38

CHAPTER 4 .................................................................................................. 39 Results .......................................................................................................... 39

4.1. Cell culture ......................................................................................... 39 4.1.1. Neonatal cell cultures .................................................................. 39

4.2. Purity of microglia in culture ............................................................... 40 4.3. Morphological alterations following activation .................................... 42

4.3.1. Morphology of activated microglia assessed by lectin staining .... 42 4.3.2. Ultrastructural changes in microglia following activation .............. 46

4.3.3. Immunophenotype of microglia following activation ........................ 50 4.3.4. Assessment of microglia proliferation following activation ............... 54

4.3.4.1. Microglia viability following activation ........................................ 54 4.3.4.2. Microglia proliferation as assessed by CFSE staining .............. 54

4.4. Adult cell culture ................................................................................. 61 4.4.1. Purity of adult microglia ............................................................... 63

CHAPTER 5 .................................................................................................. 66 Discussion .................................................................................................... 66

5.1. Primary microglia cell culture ............................................................. 66 5.2. Morphological changes following activation ....................................... 67 5.3. Ultrastructural alterations following activation .................................... 70 5.4. Immunophenotype of microglia following activation ........................... 72 5.5. Microglia proliferation following activation .......................................... 74 5.6. Establishing adult microglia cultures .................................................. 75

CHAPTER 6 .................................................................................................. 78

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Conclusion and future recommendations ................................................. 78 6.1 Conclusion .......................................................................................... 78 6.2. Future recommendations ................................................................... 79

REFERENCES…………………………………………………………………….77 APPENDICES……………………………………………………………………..90 BIODATA OF STUDENT………………………………………………………...94 LIST OF PUBLICATIONS………………………………………………….…….95

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LIST OF TABLES

Table Page

2.1 Examples of signals and modulators of microglial activation. 10

2.2 Microglial cell membrane receptors. 12

2.3 Factors released by microglia following activation. 16

2.4 Effect of aging on cells of the innate immune system. 26

3.1 Antibodies used in this study. 32

4.1

4.2

Cell yield from neonatal (3-5d) rats.

Immunophenotyping result of LPS or Aβ-treated microglia

39

49

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LIST OF FIGURES

Figure Page

2.1 Activity states of microglia in CNS. 18

4.1 Neonatal microglial cell culture as viewed under

phase-contrast microscope.

40

4.2 Histochemical staining of neonatal microglia. 42

4.3 Morphological alteration of microglia following treatment by LPS or

Aβ using fluorescent microscope.

43

4.4 Confocal images of LPS or Aβ-treated microglia. 45

4.5 TEM micrographs depicting mitochondrial alterations in LPS or Aβ-treated microglia.

47

4.6 TEM micrographs depicting endoplasmic reticulum changes in

LPS or Aβ-treated microglia.

48

4.7 TEM micrographs depicting nuclear changes in LPS or Aβ-treated

microglia.

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4.8 Immunophenotype analysis of CD40 and MHC II in untreated

microglia.

51

4.9 Immunophenotype analysis of CD40 and MHC II in LPS-treated

microglia.

52

4.10 Immunophenotype analysis of CD40 and MHC II in Aβ-treated

microglia.

53

4.11 Histogram depicting proliferation of microglia in culture. 55

4.12 Proliferation evaluation of unstained microglia. 56

4.13 Proliferation evaluation of CFSE-stained microglia on day 0. 57

4.14 Analysis of CFSE staining on untreated microglia for day 6 in 58

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culture.

4.15 Proliferation evaluation of CFSE stained in LPS-treated microglia

in day 6.

59

4.16 Proliferation evaluation of CFSE stained in Aβ-treated microglia in

day 6.

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4.17 Histogram depicting proliferation of microglia following LPS and

Aβ treatment.

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4.18 Chart depicts cell yield of adult microglia cultures following various

technical improvisations.

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4.19 Adult rat microglia cell culture as viewed by phase-contrast

microscope.

62

4.20

Histochemical staining of adult microglia.

63

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LIST OF ABBREVIATIONS

Α Alpha

Γ Gamma

CNS Central nervous system

Aβ Beta amyloid

LPS Lipopolysaccharide

AD Alzheimer’s disease

PD

MS

EM

APC

Parkinson’s Disease

Multiple sclerosis

Electron microscopy

Antigen presenting cells

RER Rough endoplasmic reticulum

SER Smooth endoplasmic reticulum

TEM Transmission electron microscopy

GM-CSF Granulocyte macrophage stimulating factor

M-CSF Macrophage colony stimulating factor

TLR Toll like receptor

Gp Glycoprotein

IL Interleukin

IFN Interferon

°C Degrees centigrade

µl Microlitre

BSA Bovine serum albumin

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EDTA Ethylene-diamine-tetra-acetic acid

g Gram

H Hours(s)

Min Minute(s)

Ml Millilitre

NO Nitric oxide

ROS Reactive oxygen species

CFSE Carboxyfluorescein diacetate succinimidyl ester

FACS Fluorescence activated cell sorting

MHC Major histocompatibility complex

CD Cluster of differentiation

FITC Fluorescein isothiocyanate

% Percentage

µg Microgram

Th T helper

U International unit

DC Dendritic cell

DNA Deoxyribonucleic acid

NFkB nuclear factor kappa beta

CHAPTER 1

INTRODUCTION

1.1. Background

Microglia are the residential macrophages of the central nervous system

(CNS). Their immunological roles include phagocytosis of invading

microorganisms and removal of dead cells within the CNS. In the normal

CNS, microglia are in a resting state, however they quickly become activated

following any stress or injury to the CNS.

Ramified microglia, represent the resting state of the cells, are found

throughout the healthy CNS. They are composed of long branching

processes and a small cellular body. While the branches of resting ramified

microglia are constantly moving and surveying the surrounding area, the cell

body remains fairly motionless. The branches are very sensitive to small

changes in physiological condition. Unlike activated or amoeboid microglia,

ramified microglia are unable to phagocytose cells (Aloisi, 2001; Christensen

et al., 2006). In addition to being very sensitive to small changes in their

environment, each microglial cell also physically surveys its domain on a

regular basis. While moving through CNS regions, the microglial cell

scavenges for foreign material, damaged cells, apoptotic cells, neural

tangles, DNA fragments, or plaques. It will then become activated and

phagocytose the material or launch inflammation if necessary. In this manner

microglial cells also act as “housekeepers”, cleaning up random cellular

debris (Aloisi, 2001).

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Following activation, microglia proliferate, express MHC class I and II

antigens, cell adhesion molecules, release inflammatory cytokines and

become phagocytotic cells (Gehrmann et al., 1995). Injury to neurones leads

to activation of microglia, which causes cellular hypertrophy, decrease in

ramification and cellular proliferation (Olesen et al., 2007).

It is believed that hyperactivation of microglia may however be counter-

productive in its effort to limit CNS damage. Activated microglia is detected

brains of patients with Alzheimer’s disease (AD) and Parkinson’s disease

(PD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), human

immunodeficiency virus (HIV) infection, and prion-related diseases (Liu &

Hong, 2003). Previously, the inflammatory activities of microglia were

conceived as only a reactive response to neuronal damage. However,

increasing reports demonstrate that inflammatory activities of microglia

worsen neuronal damage, which then fuels a self-propelling cycle of neuronal

death. Proinflammatory mediators such as the cytokines IL-6, IL-1β, and

TNF-α are secreted by activated microglia and can induce cell death in

neurodegeneration (Glezer et al., 2007). Thus, while the triggers of various

neurodegenerative diseases are different, inflammation may be a basic

mechanism driving the progressive nature of these diseases. Although

astrocytes and infiltrating lymphocytes have also been listed as contributors

to inflammation-mediated neurodegeneration, microglia are concerned as

critical components of neuroinflammation (Aloisi et al., 1998; Stoll & Jander,

1999; Miljkovic et al., 2007).

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It is of great scientific benefit to further study the process of microglia

activation and its role in degeneration. Limiting the inflammatory actions of

microglia may prove to be a suitable therapeutic approach for

neurodegenerative and neuroinflammatory diseases. In this study in vitro

responses of microglia to two activators, LPS or Aβ were determined.

Although in vivo studies are equally important for dissecting the mechanisms

of microglia activation, in vitro systems have the advantage of being a more

controlled environment for the study of cell form and function.

Microglia were cultured from neonatal rats and activated with

lipopolysaccharide (LPS) or the active fragment of beta-amyloid (Aβ). LPS is

commonly used to induce microglia activation both in vitro and in vivo and Aβ

is the misfolded protein found in senile plaques of AD patients. Morphological

changes to microglia following activation include deramification, with

microglia assuming an amoeboidal shape with a condensed cytoplasm. We

further explored morphological changes by examining changes at the

ultrastructure level. Stimulation of monocytes and macrophages by LPS is

reported (Meng & Lowell, 1997) and Aβ is used to activate microglia in vitro

into a neurotoxic phenotype distinguished by secretion of excess of

proinflammatory molecules (Floden et al., 2005).

We also investigated whether activation of microglia affects expression of the

cell surface molecules, MHC II and CD40. MHC II is expressed on antigen

presenting cells and is reportedly upregulated following microglia activation.

CD40 is a co-stimulatory molecule required for microglia activation, shown to

4

be upregulated in IFN-γ-activated microglia (Nguyen et al., 1998). Strategies

to attenuate microglia inflammatory responses by inhibiting the activation of

microglia (by suppression of CD40 expression) may be of benefit for several

neuroinflammatory diseases.

In vivo studies suggest that microglia number expand following insult via (i)

resident microglia division, and/or (ii) recruitment of circulating monocytes

and differentiation to microglia (Lawson et al., 1992; Streit, 2006; Remington

et al., 2007). Details of this proliferation wave in activated microglia are

limited. In this study, we will evaluate the in vitro proliferation cycle of

microglia following treatment with LPS or Aβ.

Majority of in vitro studies utilise microglia isolated from neonatal rodents, as

these cells have high proliferative capacity in culture. However, as neonatal

microglia are expected to respond differently from adult microglial cells

(Conde & Streit, 2006), the need for a suitable culture system for adult

microglia is also apparent. It is assumed that neonatal microglial cells are

functionally immature. Compared to the amoeboidal neonatal microglia, adult

microglia demonstrate a ramified morphology. Several studies have used ex

vivo isolated adult microglia to scrutinise the activities of these cells. The

difficulty with using adult microglial cells that have been isolated ex vivo is

their affinity to undergo cell death a few days after culture.