Experimental in vivo and in vitro models of multiple sclerosis: EAE and beyond.

Multiple Sclerosis and Related Disorders 1 (2012) 15–28

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Multiple Sclerosis and Related Disorders journal homepage: www.elsevier.com/locate/msard

Review

Experimental in vivo and in vitro models of multiple sclerosis: EAE and beyond Markus Kipp a,b, Baukje van der Star a, Daphne Y.S. Vogel a,c, Fabıola Puentes d, Paul van der Valk a, David Baker d, Sandra Amor a,d,n a

Department of Pathology, VU University Medical Centre, PO Box 7057, 1007 MB Amsterdam, The Netherlands Institute of Neuroanatomy, Faculty of Medicine, RWTH Aachen University, Aachen, Germany c Department of Molecular Cell Biology and Immunology, VU University Medical Centre, Amsterdam, The Netherlands d Neuroimmunology Unit, Blizard Institute, Queen Mary University of London, Barts and The London School of Medicine and Dentistry, London, UK b

a r t i c l e i n f o

abstract

Article history: Received 15 June 2011 Accepted 5 September 2011

Although the primary cause of multiple sclerosis (MS) is unknown, the widely accepted view is that aberrant (auto)immune responses possibly arising following infection(s) are responsible for the destructive inflammatory demyelination and neurodegeneration in the central nervous system (CNS). This notion, and the limited access of human brain tissue early in the course of MS, has led to the development of autoimmune, viral and toxin-induced demyelination animal models as well as the development of human CNS cell and organotypic brain slice cultures in an attempt to understand events in MS. The autoimmune models, collectively known as experimental autoimmune encephalomyelitis (EAE), and viral models have shaped ideas of how environmental factors may trigger inflammation, demyelination and neurodegeneration in the CNS. Understandably, these models have also heavily influenced the development of therapies targeting the inflammatory aspect of MS. Demyelination and remyelination in the absence of overt inflammation are better studied in toxin-induced demyelination models using cuprizone and lysolecithin. The paradigm shift of MS as an autoimmune disease of myelin to a neurodegenerative disease has required more appropriate models reflecting the axonal and neuronal damage. Thus, secondary progressive EAE and spastic models have been crucial to develop neuroprotective approaches. In this review the current in vivo and in vitro experimental models to examine pathological mechanisms involved in inflammation, demyelination and neuronal degeneration, as well as remyelination and repair in MS are discussed. Since this knowledge is the basis for the development of new therapeutic approaches for MS, we particularly address whether the currently available models truly reflect the human disease, and discuss perspectives to further optimise and develop more suitable experimental models to study MS. & 2011 Elsevier B.V. All rights reserved.

Keywords: EAE Virus models Cuprizone Neurodegeneration Blood–brain barrier In vitro

Contents 1. 2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Viral models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2.1. Semliki Forest virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 2.2. Theiler’s murine encephalomyelitis virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 2.3. Mouse hepatitis virus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Experimental autoimmune encephalomyelitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 3.1. History of EAE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 3.2. Disease courses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 3.3. Choice of animal and autoantigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 3.4. Models of neurodegeneration, grey matter and cortical pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 3.5. Pathogenic mechanisms in EAE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Transgenic mouse models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

n Corresponding author at: MS Research Group, Department of Pathology, VU University Medical Centre, PO Box 7057, 1007 MB Amsterdam, The Netherlands. Tel.: þ31 20 444 2898; fax: þ 31 20 4442964. E-mail address: [email protected] (S. Amor).

2211-0348/$ - see front matter & 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.msard.2011.09.002

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M. Kipp et al. / Multiple Sclerosis and Related Disorders 1 (2012) 15–28

4.1. Autoimmune response to myelin antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 4.2. Chemokines and cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 4.3. Conditional knock-out mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Toxin models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 5.1. Ethidium bromide (EB) and lysophosphatidylcholine (LPC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 5.2. The cuprizone model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 5.3. Mode of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 5.4. Which toxin-induced model should be applied? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 In vitro models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 6.1. Microglia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 6.2. Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 6.3. Oligodendrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 6.4. Astrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 6.5. Co-cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 6.6. Spheroid and 3D CNS cultures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 6.7. Brain slice cultures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 6.8. Blood–brain barrier models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Perspectives and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

1. Introduction Multiple sclerosis (MS), a common demyelinating disease of young adults can be classified in the group of inflammatory, demyelinating disease. That many inflammatory, demyelinating disorders in humans and animals, where the aetiological agent is known, are due to viral infection has lead to the development of several viral models to study MS. The autoimmune view of MS is strongly supported by the animal model experimental autoimmune encephalomyelitis (EAE), a group of disorders characterised by inflammation, myelin damage and neurodegeneration induced following immunisation with brain antigens. Remyelination, however, is better studied in toxin models such as the cuprizone model, where adaptive immune responses are not involved. Despite the extensive use of these models, the clinical course, immunology and neuropathology reflect only part of the pathological spectrum of MS, indicating that responses to therapies in animal models often cannot predict efficacy in humans. Thus, to understand the interactions between the human immune system and the human CNS, in vitro cultures of microglia, oligodendrocytes, astrocytes and neurons have been established. To study complex interactions of the blood–brain barrier (BBB) and cellular interactions, mouse brain spheroid cultures have been used to examine e.g. myelin damage. Of more relevance are human brain organotypic brain slice cultures in which the cells as well as the extracellular matrix are intact. While the latter models require further refinements to model demyelination and neurodegeneration, these new approaches highlight the importance and availability of novel yet relevant models to study the complexity of MS. Several important issues surrounding the use of experimental animal models to study MS, or indeed other disorders, have recently been addressed (Baker et al., 2011; Vesterinen et al., 2010). Here, we also draw attention to the ‘Animals in Research: Reporting In Vivo Experiments’ (ARRIVE) guidelines recently released with the aim of improving the quality of research using animals (Kilkenny et al., 2010). Given the currently available 8400 papers on EAE it is appropriate to highlight and bring to attention this report. Examining and, where appropriate, adopting the ARRIVE guidelines will clearly improve experimental findings in MS research using experimental systems to model the disease.

2. Viral models The similarities between MS pathology and viral demyelinating disorders of the CNS (Table 1) as well as epidemiological

observations have made the infectious aetiology of MS an attractive hypothesis. Electron microscopical and virological studies have supported this by revealing the presence of viruses in MS brain tissues. In animals natural infections e.g. canine distemper virus in dogs, visna virus in sheep, and mink encephalitis, induce myelin damage in the CNS, and several of these were used as models to study events in MS. However, the impracticalities of models involving large animals has thus spawned several rodent models as discussed below.

2.1. Semliki Forest virus Semliki Forest virus (SFV), an enveloped virus belonging to the Togaviridae family, was first isolated form mosquitoes in 1942 (Smithburn et al., 1946). Several strains are neurotropic in rodents and have been designated by their virulence in laboratory rodents. The common strains are the lethal L10, mutant M9 and avirulent A7; the latter two induce demyelination in adult mice. Following peripheral inoculation SFV is neuroinvasive, crosses the BBB and infects neurons and oligodendrocytes but not astrocytes. In vivo neurons, but not oligodendrocytes, rapidly downregulate virus replication, indicating that CNS cells differ in their ability to suppress SFV replication (Fragkoudis et al., 2009). In culture, infection induces dedifferentiation of mature oligodendrocytes and apoptosis (Glasgow et al., 1997). Dissection of the pathogenic mechanisms underlying demyelination in vivo has revealed that rather than direct viral-induced lysis of oligodendrocytes, CD8þ T cells are required to induce inflammatory demyelination. Neither athymic nu/nu mice nor severe combined immunodeficiency (SCID) mice develop demyelination, despite high levels of virus in the CNS (Amor et al., 1996a; Fazakerley et al., 1983). Anti-viral antibodies clear the systemic infection and control the CNS infection but lack the capacity to induce demyelination directly. This argues against autoimmunity in SFV infection, although several studies implicate molecular mimicry (Mokhtarian et al., 2003) and determinant spreading as well as expression of denovo antigens in the CNS (Verbeek et al., 2007). While a useful model to study de- and remyelination in mice as well as the impact of therapies in an infectious model (SoiluHanninen et al., 1997), SFV infection does not lead to clinical disease, probably because infection does not lead to severe axonal damage. Despite this, SFV infection is a good model to examine the impact of immunosuppressive therapies on CNS infections,


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Table 1 Virus-induced demyelinating disorders of animals. Virus (family)

Animal

Clinical

Pathology

Reference

Canine distemper (Paramyxoviridae)

Dogs Natural infection

Neurological symptoms—seizures, fits and ataxia

Vandevelde and Zurbriggen (2005)

Maedi-Visna virus (Retroviridae)

Sheep Natural infection

Measles (Paramyxoviridae) Semliki Forest virus (Togaviridae)

Rats

Fatal slow virus infection of CNS. Commonly seen in Icelandic sheep Subacute encephalomyelitis

Acute disease. Metabolic changes in oligodendrocytes. The inflammatory response is associated with demyelination in the disease progression Strong role for the immune response in the pathological lesions

Liebert et al. (1990)

TMEV (Picornviridae)

Mice Natural infection

Inflammatory demyelination. Role for autoimmunity to myelin proteins Demyelination in spinal cord, brain and optic associated with inflammation. Myelin loss is associated with CD8 T cells In sub-lethal strains viral infection of neurons is cleared but followed by inflammation and myelin damage due to CD8 T cell attack

Mouse hepatitis virus (Coronaviridae)

Rats, mice

Rats, mice

Avirulent A7(74) injected i.p. induces subclinical disease Flaccid hind limb paralysis. Fatal encephalitis or biphasic disease with sub-lethal strains Paralysis following i.c. and i.n. inoculation

Replication in neurons, ependyma cells, astrocytes, oligodendrocytes and microglia. Demyelination in part due to direct viral destruction of cells but also due to specific immune attack

Benavides et al. (2009)

Amor et al. (1996a) Fazakerley and Walker (2003) Fazakerley and Walker (2003) Tsunoda and Fujinami (2010) Fazakerley and Walker (2003)

a crucial point given the unexpected emergence of infections with several immunosuppressive approaches in humans.

CNS. This prevents the study of how peripheral infections induce autoimmunity in the CNS.

2.2. Theiler’s murine encephalomyelitis virus

2.3. Mouse hepatitis virus

Theiler’s murine encephalomyelitis virus (TMEV) is a singlestranded RNA virus belonging to the family of Picornaviridae. Although an enteric mouse pathogen, TMEV was first identified by Max Theiler to cause paralysis and encephalomyelitis in mice (Theiler, 1934). TMEV can be divided into two main groups: the highly neurovirulent viruses that induce fatal encephalitis and the low-neurovirulent viruses including Daniels (DA) and BeAn strains. Susceptibility to infection is major histocompatability complex (MHC) class I dependent. Infection with the low-neurovirulent strains induces a biphasic infection in mice consisting of poliomyelitis in which virus replicates in grey matter of the CNS. The virus is cleared to very low levels within 2 weeks. This episode is followed by a persistent infection associated with inflammation in the CNS and chronic inflammatory demyelination. The chronicity of disease depends on the mouse strain that determines the extent of disease progression, axonal damage and spinal cord atrophy. Studies show that remyelination is sparse in some mouse strains (Tsunoda and Fujinami, 2010). Several pathological mechanisms contribute to myelin damage in TMEV infection. MHC class I molecules determine susceptibility to infection and, thus, the extent of myelin damage. However, demyelination is also observed in athymic nu/nu mice, indicating direct viral damage to myelin. In SJL mice myelin damage is immunologically mediated by CD4þ cells directed to myelin antigens. The anti-myelin autoimmune responses arise via epitope spreading during the chronic stage of disease or due to molecular mimicry. In vitro, TMEV infects immature oligodendrocytes and blocks cell differentiation (Pringproa et al., 2010) indicating that infection could delay or inhibit remyelination in vivo and that demyelination maybe due to direct oligodendrocyte killing. The virus persists in oligodendrocytes and microglia in the spinal cord leading to atrophy as a result of loss of the small and large diameter axons. Mechanisms involving perforin-dependent effector cells contribute to axonal damage have been suggested to be dependent upon but separable from demyelination (Howe et al., 2007). While very useful to study virus-induced myelin and axonal damage, a disadvantage of the TMEV model is that disease only occurs after direct injection of the virus into the

Mouse hepatitis virus (MHV) is a positive-stranded RNA virus of the Coronaviridae family and a natural pathogen of mice, inducing neurological disease as well as gastrointestinal and respiratory disorders. Similar to TMEV, the pathogenicity of MHV is dependent on the viral strain, mouse host strain and inoculation route. Following intracranial inoculation with the neurovirulent strain MHV-A59, susceptible mice develop acute encephalitis in which the virus infects astrocytes, oligodendrocytes and microglia in the brain, spinal cord and optic nerve (Shindler et al., 2008). Infection leads to increased innate immune responses including interferons which are protective in disease. NK cells and microglia contribute to inflammation, but do not play a major role in viral clearance. Neutralising antibodies do not aid viral clearance in acute disease, but are thought to contribute to the low viral loads observed in chronic disease. In acute disease, CD8þ T cells, the predominant cell type contributing to viral clearance in the CNS, also contribute to the viral persistence in the chronic phase. Although virus infection contributes to myelin damage by inducing oligodendrocyte apoptosis, the main effectors of demyelination are T cells and macrophages. Axonal damage is also a pathological feature of MHV infection and several mechanisms, including immune-mediated attack have been suggested (Dandekar et al., 2001; Das Sarma et al., 2009) clearly contributing to the usefulness of this model to study MS.

3. Experimental autoimmune encephalomyelitis Immunisation of susceptible animals with CNS antigens gives rise to a spectrum of inflammatory disorders collectively named EAE (Table 2). Although the experimental disease in animals was originally termed experimental disseminated encephalomyelitis, the idea that the ‘disease’ was allergic gave rise to the name experimental allergic encephalomyelitis. More recently, allergic has been replaced by autoimmune. Despite differences in disease course and pathology, EAE is still the most intensely used experimental model of MS. As well as a model for MS, EAE studies

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Table 2 Spectrum of EAE. EAE

Animal

Clinical

Pathology

Reference

Hyperacute

Rats Adoptive transfer of lymphocytes after SCH immunisation Biozzi ABH mouse Immunisation with MAG or MBP in CFA with PT Lewis rats Immunisation with MBP in CFA Rhesus monkeys Oligodendrocyte specific protein in CFA MOG 35–55 in C57BL/6 or Biozzi ABH mice in CFAþ PT Marmoset rMOG in CFA; native MOG in myelin in CFA Biozzi mouse Immunisation with rMOG, MOG 8–21 PLP, PLP 56–70 or SCH in CFA/CFAþ PT

Hyperacute EAE one day after transfer and PT

Deposits of fibrin and neutrophils

Levine and Sowinski (1973)

Acute monophasic disease

Minimal demyelination

Amor et al. (1996b), Morris-Downes et al. (2002b)

Acute monophasic disease

Minimal demyelination

Swanborg (2001)

Optic neuritis

Demyelination and inflammation in the optic nerve

Bajramovic et al. (2008)

Chronic disease no or very infrequent recovery Chronic demyelinating

Extensive neuronal loss associated with inflammation in the spinal cord Demyelination in CNS

Smith et al. (2005)

Chronic relapsing

Minimal demyelination in acute EAE and more extensive in relapses. Axonal damage and neurological deficit increases with time and number of relapses Mainly inflammatory with varying degrees of myelin damage

Amor et al. (1993), Amor et al. (1994), Amor et al. (1996b), Baker et al. (1990)

Inflammation and extensive demyelination with remyelination in relapse EAE Marked gliosis, demyelination, remyelination and axonal and neuronal loss Inflammation in CNS, limited demyelination Inflammation and demyelination in spinal cord and optic nerve Axonal loss, grey matter damage and myelin damage

Gambi et al. (1989) Raine et al. (1978)

Acute

Acute EAE Clinical optic neuritis Chronic Chronic

Chronic relapsing

Chronic relapsing

Chronic relapsing

Chronic relapsing

Dark agouti rats Immunisation with rMOG, SCH in IFA Guinea pig Hartley and Strain 13

Secondary progressive

ABH mice Immunisation with SCH in CFA

Secondary progressive EAE

Spontaneous

Humanised double transgenic mice TCR for MBP and HLA-DR15 Humanised double transgenic mice MOG TCR and HLA-DR15 ABH mice Immunisation with NF-L in CFAþPT

Monophasic with severe paralysis Monophasic

Spontaneous Spastic paresis

Acute and chronic relapsing

Spastic paresis and paralysis

have provided important contributions to our understanding of neuro-immune interactions within the CNS. 3.1. History of EAE Probably the first recorded, yet unintentional induction of experimentally induced encephalomyelitis was carried out by Louis Pasteur in 1885, while developing a rabies vaccine. Between 1894 and 1914, rabies virus was propagated in rabbits, and a vaccine was produced from spinal cord tissues. The vaccine, along with residual CNS tissues was repeatedly injected to protect against rabies. Some preparations, however, induced paralysis, and in the most severe forms mortality in vaccinated subjects. Pathological examination of the CNS revealed extensive inflammation and perivenous demyelination in the brain and spinal cord. It was these early human studies (Baxter, 2007) that stimulated Thomas Rivers’ studies in rhesus monkeys, from which parallels were drawn with human paralytic inflammatory demyelinating diseases (Rivers and Schwentker, 1935). While Rivers’ studies required repeated injections of brain tissues Kabat, who first likened EAE to MS, showed that augmenting the immune response by using an adjuvant developed by Freund, only a single injection was required to induce disease. This protocol had the added advantage that disease occurred within weeks rather than months (Kabat et al., 1946). The adjuvant was originally thought to act by boosting antibody responses. It is now known that the adjuvant elicits Th1 and Th2 responses via Toll-like receptor activation. Notably for chronic relapsing disease in the DA rat, addition of mycobacterium to the adjuvant is not required (Lorentzen et al., 1995).

Genain and Hauser (2001) and Jagessar et al. (2008)

Lorentzen et al. (1995) Swanborg (2001)

Hampton et al. (2008)

Ellmerich et al. (2005) Bettelli et al. (2003) Huizinga et al. (2007) Huizinga et al. (2008)

3.2. Disease courses Immunisation of susceptible animals with CNS tissues and adjuvant elicits either a monophasic neurological episode of paralysis, from which the animals recover and are refractory to reinduction of disease, or chronic paralysis from which the animals do not recover. More relevant to MS are the models which exhibit chronic-relapsing neurological episodes and progressive disability (Table 2). EAE induced by immunisation is referred to as actively induced EAE. Alternatively, EAE can be induced following adoptive transfer to naive recipients of lymph node cells, or specific T cell lines and clones (both CD4þ and CD8þ) from immunised animals. This is termed adoptive transfer or passive EAE. Whatever the induction regimen, the initial phase of disease is usually termed the acute phase (Fig. 1A), and correlates with the mononuclear cell infiltrates in the CNS. In species and strains where the animals recover, this recovery period is referred to as remission. If animals do not recover the disease is referred to as chronic EAE (Fig. 1B). To increase the susceptibility Bordetella pertussis toxin may also be injected, but this may induce hyperacute EAE (Table 2) (Levine and Sowinski, 1973). The exact mechanism by which pertussis toxin promotes EAE has not been fully elucidated, although non-selective expansion of T cells and vascular changes in the CNS have been suggested (Richard et al., 2011). Several animals, particularly strain 13 guinea pigs, DA rats, SJL and ABH mice exhibit recurrent phases of neurological deficit (Fig. 1C). In the relapse phase myelin damage and axonal loss is more prominent than in the acute stage (Amor et al., 1994; Baker


demyelination, axonal and neuronal loss, and marked gliosis, all features of chronic MS (Hampton et al., 2008; Al-Izki et al., 2011 this issue). This model is thus ideal to investigate the pathological mechanisms underlying disease progression and neurodegeneration as well as the development of therapies for neuroprotection.

clinical score

4 3 2

3.3. Choice of animal and autoantigen

1 0

Acute

10

Remission

20

30

40

50

60

70

50

60

70

clinical score

4 Chronic

3 2 1 0 10

clinical score

4

30

Remission

40

Remission

Secondary progressive

2 1 Acute

0 10

clinical score

20

3

4

20

2 Relapse

1 Relapse

30

40

50

60

70

Anti-myelin antibody augmented acute EAE

3 2 1 0

19

Acute

18 14 10 12 Post sensitisation day Fig. 1. Clinical courses of EAE. Immunisation of animals with myelin antigens gives rise to a spectrum of EAE in which the clinical scores represent the neurological deficit observed. The scoring system varies but commonly ranges from 0 representing control or no clinical disease to 4 representing severe clinical paralysis commonly observed by flaccid paralysis of the hind limbs. For ethical reasons score 4 is generally the most severe score although in some reports score 5 is used to represent moribund or death from EAE. Immunisation gives rise to an acute phase (A) in which animals recover (remission) yet do not develop further episodes of disease. In some cases animals do not recover from the acute phase and the disease is referred to as chronic EAE (B). In those models where animals recover, the remission phase is followed by relapse phases (C). In this case the animals develop subsequent relapses in which neurological deficit accumulates and eventually do not return to baseline (0) when the animals enter the secondary progressive EAE phase. (D) Clinical course of acute EAE in ABH mice in which pertussis toxin has been omitted (black line, note the reduced clinical score compared to A) do not show extensive demyelination (E, LFB staining). To augment myelin damage injection of antibodies to myelin oligodendrocyte glycoprotein at the onset of EAE (day 10) exacerbates clinical disease (D, dotted line) concomitantly with myelin loss (F, arrow points to remnants of myelin, LFB staining).

et al., 1990; Raine et al., 1978). An additional advantage of chronic relapsing EAE in the ABH mouse is the development of secondary progressive disease. The pathology of these mice shows extensive

While many animal species and strains, (Table 3) including humans are susceptible to EAE the availability of many biological tools to probe the disease and the availability of genetically engineered animals makes the mouse ideal as a host for an experimental model of MS. Initially, mice only developed monophasic EAE, but by adapting the protocol and changing the antigen, relapsing EAE could be induced following active sensitisation or T cell transfer (Pettinelli and McFarlin, 1981; Zamvil et al., 1985). Initially, because of the ease in purification and the quantities available, myelin basic protein (MBP) was the most widely-used antigen, and studies were largely restricted to SJL (H-2s) and PL/J (H-2u) mice. While much more difficult to produce, proteolipid protein (PLP) has also been used although the use of immunodominant peptide epitopes of PLP are preferred due to the hydrophobicity of the protein (Amor et al., 1993; Tuohy et al., 1989) (Table 3). The discovery that the CNS specific protein myelin oligodendrocyte glycoprotein (MOG) was also encephalitogenic in SJL and ABH (H-2dq1) mice (Amor et al., 1994) allowed the development of the MOG model in C57BL/6 (H-2b) (Mendel et al., 1995). This was a crucial step in MS and EAE research since the majority of the transgenic mice available are generated on the C57BL/6 background, allowing detailed study of the disease processes. The disadvantage is that only a single peptide epitope of MOG i.e. MOG35–55 induces EAE in this strain and, whatever the strain or species, this peptide induces a severe chronic EAE disease course (Smith et al., 2005). This is intriguing and may very well explain some MS phenotypes, but it is crucial that other mouse strains are studied to prevent bias. While MOG35–55 induces a chronic disease in rats, mice, rhesus monkeys and marmosets, the use of recombinant MOG has lead to the development of several relapsing-remitting models that better model the clinical and pathological aspects of MS. To examine the role of post-translational modification in disease, native MOG-in-myelin has been used for EAE induction (Amor et al., 1994; Smith et al., 2005). These studies revealed that T-cell responses to native MOG-in-myelin were responsible for triggering demyelination and chronic-relapsing disease. Moreover, that antibody to MOG but not other myelin proteins, augmented EAE and demyelination suggested a role for humoral responses as well (Morris-Downes et al. 2002a). Similar to MS, age, gender and environmental factors have a profound influence on disease susceptibility, severity and course of EAE. For example young male SJL mice immunised with PLP are relatively resistant to EAE whereas older males and female SJL mice of any age are susceptible. Young C57BL/6 mice and Wistar rats develop acute EAE and remission whereas middle-age males developed severe chronic EAE (Ditamo et al., 2005; Matejuk et al., 2005). As in MS pregnant females show a reduced susceptibility to disease, probably as a result of immune suppression (Evron et al., 1984). These findings, along with the observation that older mice immunised in winter are more susceptible to EAE indicate an influence of genetics, gender and age in disease susceptibility. 3.4. Models of neurodegeneration, grey matter and cortical pathology Animals with EAE also develop cortical and deep grey matter demyelination (MacKenzie-Graham et al., 2006; Mangiardi et al.,

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Table 3 Encephalitogenic peptide epitopes of MBP, PLP and MOG in mice. Mouse

MHC class II

MBP

PLP

MOG

Biozzi ABH SJL PLJ C57BL/6 NOD BALB/c SWR DBA/1 BIO. RI I 1 A.CR

g7 s u b g7 d q q r f

1–12 17–27, 89–101 Ac1-9, 33–35 nd 1–12 nd 87–99 nd 89–101 Acl-11, 87–99, and 9–20

56–72 139–151, 178–191 43–64 nd 57–72 56–72 103–116,141–149 nd nd nd

8–21, 35–55, 43–57 92–106 35–55 35–55 8–21, 35–55, nd nd 79–96 nd nd

Humanised transgenic mice Ob.1112 Line 7 Hy.2E11 MH2-C38 2D1

HLA-DR2a HLA-DR2b HLA-DR4 HLA-A3

85–99 85–99 111–129 45–53

nd—not determined, MOG—myelin oligodendrocyte glycoprotein, MAG—myelin basic protein, PLP—proteolipid protein.

2011; Pomeroy et al., 2008). In addition, decreased levels of antioxidants and increased glutamate/glutamine levels (Castegna et al., 2011) as well as down-regulation of genes involved in mitochondrial function are observed in brain tissues of mice with EAE (Zeis et al., 2008). Contrary to the spinal cord there is little evidence of immune/inflammatory cell invasion in the brain suggesting that the pathological changes observed in the brain are probably due to inflammation elsewhere in the CNS. To specifically induce lesions in the cortex Mangiardi and colleagues’ steriotactically injected TNFa and IFNg in the cerebral cortex of animals with subclinical EAE (Mangiardi et al., 2011). These studies revealed that while either cytokine alone was pathogenic coinjection of both TNFa and IFNg was necessary to induce focal lesions of myelin damage. Similar to myelin, autoimmunity to axons and neurons may also contribute to disease. Immunisation with neurofilament light, an axonal cytoskeleton protein, or passive transfer of antibodies to neurofascin and contactin-2 induces and augments neuronal damage and axonal degeneration (Derfuss et al., 2009; Huizinga et al., 2007; Huizinga et al., 2008; Mathey et al., 2007). Neuronal damage also occurs after immunisation with myelin antigens, and recent studies show that such axonal damage invoked in EAE is in part reversible (Nikic et al., 2011). Extensive axonal damage is also observed in the secondary-progressive EAE model in ABH mice immunised with whole spinal cord homogenate (Hampton et al., 2008). 3.5. Pathogenic mechanisms in EAE As discussed above, T cells, particularly Th1 CD4 þ cells are classically reported to transfer EAE and thus play a crucial role in the pathogenesis of EAE. Depleting CD4 þ cells or therapies inhibiting MHC class II interactions block the induction phase and severely reduce clinical relapses. Yet several studies show that Th2 cells as well as CD8þ cells induce EAE, an important finding since CD8þ T cells dominate inflammatory MS lesions (Johnson et al., 2010). While EAE cannot be induced by B cell transfer, the role of B cells in antigen presentation, regulation or antibody production remains to be fully clarified. Antibodies to myelin and neuronal antigens are pathogenic since they augment demyelination and neuronal damage in mice; although it is still unclear how these autoantibodies induce damage. Autoimmunity is clearly necessary for acute neurological disease while immune regulation and repair of the BBB induces remission. It is still unclear why some EAE animals develop relapses while others exhibit chronic neurological disease.

Broadening of the autoimmune response, so called determinant spreading may be involved although this is debatable (Heijmans et al., 2005; Pryce et al., 2005; Smith et al., 2005).

4. Transgenic mouse models The generation of transgenic (tg) mice has greatly aided our understanding of the pathogenic mechanisms operating in EAE. Here we briefly review the major approaches using tg mice to examine the role of autoimmune responses to myelin, chemokines, cytokines and mechanisms of neuronal or oligodendrocyte damage. Transgenic mice expressing HLA haplotypes linked to susceptibility to MS have allowed dissection of human elements involved in EAE. While these animals offer unique approaches to understand pathogenic mechanisms redundancy in biological systems may lead to unexpected results. 4.1. Autoimmune response to myelin antigens The role of autoimmune responses to myelin antigens in EAE can be examined using CNS protein deficient mice e.g. MOG knock-out mice or T cell receptor (TCR) tg mice in which cells are directed to a particular myelin peptide known to induce EAE. MOG-deficient mice for example do not develop chronic relapsing demyelinating EAE. In addition immunisation of wild-type mice with myelin lacking MOG induces only acute disease. These findings demonstrate that autoimmunity to MOG is required for chronic EAE. In contrast, mice deficient in the myelin-associated protein alpha B crystallin, a small heat shock protein expressed in MS lesions, not only develop more severe disease but show enhanced oligodendrocyte apoptosis indicating that this protein may be protective (Ousman et al., 2007). That EAE can be induced by CD4 þ T cells directed to MBPac1-9 in PL/J (H-2u) mice led to the generation of the first tg mouse expressing the TCR specific for MBPac1-9. In later studies, TCR tg were generated from CD4þ cells directed to PLP139–151 in SJL mice and CD4þ cells directed to MOG35–55 in C57BL/6 mice (Scheikl et al., 2010). Spontaneous EAE observed in these tg mice suggested that environmental factors contributed to autoimmune-mediated, neurological disease. To examine the human component ‘humanised mice’ expressing the MBP84–102 HLA-DR2a specific TCR, MBP111–129 HLA-DR4 TCR or HLA-A3; all HLA haplotypes linked to susceptibility to MS. As discussed above, B cells are also important for induction of myelin damage. Thus, mice expressing a TCR directed to MOG35–55 (2D2 mice) were crossed with mice in which the


MOG specific immunoglobulin heavy chain was knocked-in (IgHMOG mice). In these mice 20–30% of the B cells express the B cell receptor to MOG. EAE in the double tg mice was associated with demyelination due to the presence of the MOG antibodies. Myelin damage was not observed in the 2D2 mice since these mice did not produce anti-MOG antibodies, despite the presence of T cells to MOG (Krishnamoorthy et al., 2006). 4.2. Chemokines and cytokines In both MS and EAE the contribution of chemokines and cytokines are crucial for cell recruitment and the pathogenicity of the immune responses. The role of these mediators have been dissected in tg mice, in which cytokine or chemokine genes were removed (knocked-out) or overexpressed in CNS cells. For example, overexpression of TNFa in neurons, astrocytes or oligodendrocytes enhances EAE. Likewise, overexpression of IFNg, IL-12, IL-6 and IL-3 in the CNS enhances severe neurological effects. In EAE recruitment of T cells, B cells and macrophages into the CNS is controlled by a plethora of chemokines and their ligands. Various tg mice have been generated that express chemokines and receptors in different CNS by using cell-specific promoters. However, of the many tg mice few develop clinical disease despite recruitment of cells into the CNS indicating that induction of chronic demyelinating EAE disease is multifactorial.

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toxin administration. In addition are models where the toxin is dispensed into the subarachnoid space (Guazzo, 2005; Reynolds et al., 1996). Furthermore, so called ‘‘two-hit’’ models are available. Sasaki and colleagues have described a combined model with systemic immunisation using the extracellular domain of recombinant rat MOG in incomplete Freund’s adjuvant to induce a clinically silent humoral response. Vascular endothelial growth factor was then microinjected into the spinal cord to induce a transient, focal breakdown of the BBB (Sasaki et al., 2010). Tourdias et al. (2011) induced restricted central nervous system demyelination by focal injection of TNFa and IFNg. A focal EAE model of cortical demyelination using MOG immunisation has also been reported (Merkler et al., 2006). A ‘‘three-hit model’’ was just recently introduced by Serres and colleagues. After silent immunisation with MOG, focal lesions were created by injection of TNFa and IFNg into the rat corpus callosum. Four weeks after intracerebral cytokine injection, at a point when immunohistochemical and MRI-detectable changes had returned to baseline, animals were injected intraperitoneally with lipopolysaccharide endotoxin (Serres et al., 2009). The authors found a greater inflammatory infiltrate along with more pronounced demyelination in endotoxin-reactivated lesions. These models are best viewed as complementary to one another. Despite their limitations, the rational utilisation and application of these models to address specific research questions will remain one of the most useful tools in studies of human demyelinating disorders.

4.3. Conditional knock-out mice Many tg mice express or lack the selected gene from birth, making it difficult to dissect the impact of the gene on the disease in adult mice, particularly so if lacking the gene is lethal. To overcome this, Cre loxP systems to conditionally delete genes have been applied. As an example Pohl et al. (2011) generated a double tg mouse from a mouse carrying a tamoxifen-inducible version of Cre (CreERT2), under PLP gene transcriptional control (Leone et al., 2003) by crossing it with a mouse containing a credependent diphtheria toxin-A expression cassette. Injection of adult double tg mice with tamoxifen induced cre-dependent oligodendrocyte death and axonal damage as a result of demyelination. Thus demyelination and axonal damage can be induced in the absence of an adaptive immune response and may provide an alternative to the toxin models discussed below.

5. Toxin models In principal, toxin demyelination can be induced by either focal application or systemic administration of the toxin. Agents for focal demyelination used so far are lysolecithin, also called lysophosphatidylcholine (LPC), ethidium bromide (EB) (Franklin et al., 1993; Mothe and Tator, 2008; Talbott et al., 2006), 6-aminonicotinamide (Blakemore, 1978), antibodies to oligodendrocyte-related proteins (Rosenbluth et al., 2003; Rosenbluth and Schiff, 2009), bacterial endotoxin (Felts et al., 2005) and electrolytes (Rojiani et al., 1994). Phototargeted ablation of oligodendrocytes has been reported as well (Vanderluit et al., 2000). Furthermore, pathogenic agents can be locally applied to induce demyelinated and inflamed foci. Matyszak and Perry found that the injection of killed bacillus Calmette–Gue´rin (BCG) directly into the hippocampus produces an acute inflammatory response but does not result in myelin loss. However, when animals are subsequently given BCG peripherally, a delayed-type hypersensitivity develops, associated with myelin loss (Matyszak and Perry, 1995). Feeding animals with the copper-chelator cuprizone is the most commonly used demyelination model utilising systemic

5.1. Ethidium bromide (EB) and lysophosphatidylcholine (LPC) The most commonly used toxins for focal demyelination are EB and LPC. Both agents have been used to induce demyelination in certain brain areas such as the striatum (Degaonkar et al., 2002), hippocampal formation (Goudarzvand et al., 2010), spinal cord tissue (Ousman and David, 2000; Triarhou and Herndon, 1986), optic nerve (Carroll et al., 1983), ventral roots (Smith et al., 1982), caudal cerebellar peduncles (Penderis et al., 2003) or corpus callosum (Jablonska et al., 2010). Demyelination can also be induced in the peripheral nervous system (Hall, 1973). A major difference in the pathology induced by the different toxins is the extent of astrocyte loss and the dynamics of myelin breakdown (Blakemore and Franklin, 2008). Pronounced astrocyte depletion is evident after EB-induced focal demyelination. It is, thus, possible to recreate a normal or novel astrocyte environment when combined with radiation (Franklin et al., 1993). By comparing LPC- with EB-induced lesions, it is obvious that astrocytes influence remyelination (Blakemore et al., 2002). A common formulation is 1% LPC or 0.01–1% EB in sterile 0.9% saline or PBS. Using the same volume and concentration, EB-induced lesions are much larger than LPC-induced ones (Blakemore and Franklin, 2008). 5.2. The cuprizone model Cuprizone ingestion in mice induces highly reproducible demyelination of certain brain regions, among them the corpus callosum. This commissural white matter tract represents the most frequently investigated region in this animal model (Kipp et al., 2009). After 5–6 weeks of cuprizone treatment, the corpus callosum is almost completely demyelinated, a process called ‘‘acute demyelination’’. Acute demyelination is followed by spontaneous remyelination during subsequent weeks when mice are fed normally. In contrast, remyelination is highly impaired/ delayed when cuprizone administration is prolonged (12–13 weeks or even longer), a process called ‘‘chronic demyelination’’. During late stages of acute demyelination, spontaneous remyelination

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occurs partially but it fails under a continued cuprizone challenge (Kipp et al., 2009). The first experiments using cuprizone as a toxin compound date back to the late 1960s, and were conducted by Carlton (1969). He reported that oxalic biscyclohexylidenehydrazide, a chelator used as a reagent for copper analysis, induces microscopic lesions in the brain accompanied by oedema, hydrocephalus, demyelination and astrogliosis. Based on his findings, most laboratories use 6–9-week-old mice and feeding with a diet containing 0.2–0.3% cuprizone. To assure proper demyelination in aged animals, a higher cuprizone concentration is required (Norkute et al., 2009). Furthermore, strain-dependent susceptibility to cuprizone has been reported. For example, SJL mice display a unique pattern of demyelination that does not follow the profile as seen in C57BL/6 mice. SJL mice do not readily demyelinate at the midline within the corpus callosum but show greater demyelination directly lateral to midline (Taylor et al., 2009). 5.3. Mode of action The mode of action of toxin-mediated demyelination is still debated. LPC disrupts membranes, including myelin, by inserting into lipid bilayers to form micelles (Gregson, 1989). It has also been shown to be a chemoattractant (Quinn et al., 1988) and induces vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression in arterial endothelial cells (Kume et al., 1992) and in the spinal cord (Ousman and David, 2000). EB kills oligodendrocytes and astrocytes as an intercalating dye. The mechanism of demyelination following injection of the endotoxin lipopolysaccharide (LPS) is unknown, but several possibilities can be considered. LPS is amphiphilic and could act like a detergent, directly solubilising oligodendrocyte membranes. It is also possible that the LPS-induced inflammation activates a latent viral infection, leading to destruction of oligodendrocytes. Furthermore, oligodendrocytes might be killed by factors produced by activated inflammatory cells, particularly in light of the finding that LPS-activated microglia are capable of inducing oligodendrocyte damage in culture (Lehnardt et al., 2002). The underlying mechanisms of cuprizone-induced oligodendrocyte cell death are also not fully understood. It is widelyassumed that a cuprizone-induced copper deficit might be detrimental to mitochondrial function in the brain (Venturini, 1973) and that a disturbance of energy metabolism in oligodendroglia and cell function leads to demyelination (Kipp et al., 2009; Matsushima and Morell, 2001). Why oligodendroglia should be preferentially susceptible to copper deficit is not known. It is conceivable that these cells have to maintain a vast expanse of myelin and this extraordinary metabolic demand places it in jeopardy if the demand cannot be met. Recent findings from our lab, however, challenge this concept. These show that perineuronal satellite oligodendrocytes are equally vulnerable to cuprizone-induce demyelination as compared to myelinating oligodendrocytes in the cortex (our unpublished observation). The molecular details of the mode of action of cuprizone and other commonly applied toxins remains to be fully elucidated.

require special equipment. The only drawback of this model is that the chow should be refreshed daily because animals tend to disperse it over the entire cage. Attempts to press the cuprizone powder into pellets were unsuccessful in our hands, probably due to a transient increase of temperature during the pressing procedure (our unpublished observation). The most important issue dictating the choice for any of the numerous available toxin-mediated animal models is the scientific hypothesis under investigation. If underlying mechanisms of peripheral cell recruitment into the central nervous system are of interest, one should not use the cuprizone model. Although some have claimed peripheral monocyte recruitment to occur in the cuprizone model (McMahon et al., 2002; Remington et al., 2007), results from Mildner et al. (2007) demonstrate that bone marrowderived monocytes do not contribute to the accumulation of CD11bþ cells during cuprizone-induced demyelination. These authors suggest that a priming effect of the brain due to irradiation during the experimental procedure is essential for peripheral monocyte recruitment during cuprizone induced demyelination. Furthermore, significant T cell recruitment cannot be observed in this model, probably due to direct cuprizone-mediated suppression of T cell function (Emerson et al., 2001). In contrast, peripheral leucocyte recruitment is evident in EB and LPCinduced demyelination (Ousman and David, 2000). These authors investigated cellular recruitment during LPC-induced demyelination in the spinal cord of BALB/c mice and showed early recruitment of CD4þ/CD8þ T cells and neutrophils (6–24 h post injection). At later time points, LPC induces an increase in the number of activated CD11bþ macrophages that displayed a variety of morphologies in the white and grey matter. As mentioned above, LPC-induced demyelination is due to a direct attack on the nerves myelin sheath. It, thus, occurs rapidly, and more or less independent from other factors such as genetic background of the animal. Thus, investigating remyelination in genetically modified animals is relatively straight forward. Since the same extent of demyelination in experimental groups is a sine qua non in all remyelination studies, cuprizone-induced oligodendrocyte loss and, thus, demyelination should be identical in genetically modified and wild-type animals if subsequent remyelination studies are performed. Since this is not always the case (Jha et al., 2010; Raasch et al., 2011) remyelination studies would require a conditional knock-out approach.

6. In vitro models Although animal studies have significantly contributed to the understanding of MS they only partially mimic the underlying pathogenic processes. In vitro approaches are thus crucial to study the role of CNS cells, allowing manipulations that cannot easily be performed in vivo. Many of the CNS culture systems commonly used are of animal origin. We would like to stress at this point that several essential differences exist between the rodent and human brain. Thus human in vitro models, such as human brain slice cultures or human oligodendrocytes are desired. 6.1. Microglia

5.4. Which toxin-induced model should be applied? The availability of specific technical equipment may help decide for either toxin-induced demyelination model. Focal models require a stereotactic frame as well as a micro-pump. The latter is indispensable to avoid tissue damage due to a high local pressure during the application procedure. In contrast, cuprizoneinduced demyelination is very easy to perform and does not

Microglia, the resident macrophages of the CNS constantly survey the brain parenchyma and respond rapidly to changes in the microenvironment by assuming an activated state, characterised by changes in morphology, biochemistry and function. On the morphological level, ramified microglia with thin processes can be distinguished from amoeboid microglia, characterised by short processes and a prominent cell body. Microglia activation is


a key factor in the defence of the neural parenchyma against infectious diseases, inflammation, trauma, ischaemia, brain tumours and neurodegeneration (Kreutzberg, 1996). Microglia have different roles in neurodegeneration, being benign, protective or detrimental (Napoli and Neumann, 2010; Neumann et al., 2009; Perry et al., 2010), dependent on the way they are activated. Microglia can be isolated from several species including mice (De Haas et al., 2007), rats (Skuljec et al., 2011), rhesus monkeys (Zuiderwijk-Sick et al., 2007) and humans (De Groot et al., 2001; Gibbons and Dragunow, 2010). To overcome the low cell yields microglia cell lines are available (De Vries and Boullerne, 2010) and can also be generated from mouse embryonic stem cells (Napoli et al., 2009). Microglia can be obtained from tissues by several methods, including percoll gradients (Ford et al., 1995), nutritional deprivation (Hao et al., 1991) or by collecting floating cells (Ganter et al., 1992). The most popular protocol is the shaking method described simultaneously by Giulian and Baker (1986) for rats and Frei et al. (1986) for mice. With this method, microglial cells are separated from confluent primary mixed glial cultures from newborn rodent cerebral cortex by agitation on a rotary shaker. This method allows for the preparation of highly enriched ( 495%) microglial cultures. The basic principle is that astrocytes strongly adhere to the flask and do not detach. However, a more or less 100% confluent cell layer is critical to avoid detachment of astrocytes and, thus, astrocytic contamination of the microglia culture may occur. The second principle is that oligodendrocyte progenitors adhere to the astrocyte monolayer, and thus also do not detach (Kim et al., 2011). Finally the strong adherence of microglia compared to astrocytes and oligodendrocytes to plastic surfaces allows further purification of primary microglia (Giulian and Baker, 1986). Human microglia can be isolated post mortem, from surgical material or from foetal brain, which raises ethical considerations. Since primary microglia tend to grow slowly and do not proliferate easily immortalised human microglia cell lines have been created. One example is the HMO6 cell line generated from human foetal brain using a retroviral vector encoding v-myc. This cell line expresses antigens specific for microglia/macrophages and also has a comparable cytokine gene regulation profile (Nagai et al., 2001). 6.2. Neurons Diverse sources of primary neuronal cell cultures are widely available and include primary rodent embryonal hippocampus (Bertrand et al., 2011; Copray et al., 2006) and cortex (Lorenz et al., 2009) neuronal cultures, as well as those derived from pluripotent stem cells from rats (Kitazawa and Shimizu, 2005) and rhesus monkeys (Salli et al., 2004). While human primary neurons are difficult to maintain, several studies describe systems to generate primary neuronal cultures from human tissues removed during surgery or post mortem (Brewer et al., 2001; Cano-Abad et al., 2011; Huang et al., 2008). Given the difficulty to generate primary cells from humans, several neuronal cell lines have been generated, although they also have their limitations in dendrite outgrowth and expression of mature neuronal markers. This can be overcome by differentiation strategies. For example, the neuroblastoma cell lines SH-SY-5Y cell line (Agholme et al., 2010; Forsby et al., 2009) can be differentiated with retinoic acid. In comparison, the HCN (Zhang et al., 1994) and the NT2 cell line (Pleasure et al., 1992) can be differentiated with brain-derived growth factor but despite this, they fail to express all the markers of mature cells. Another drawback of the HCN cell line is slow proliferation requiring expensive growth factors, although the

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prolonged dendrites make it very suitable to study axonal growth and damage in detail. Finally, primary neuronal-like cells have been differentiated from human stem cells (Dromard et al., 2008). Thus, despite the different caveats several neuronal cell lines are available to study mechanisms of axonal damage in MS. 6.3. Oligodendrocytes Oligodendrocytes are the myelin-forming cells, present in the CNS as pre-progenitor, O2A progenitor cell, pro-oligodendrocyte, immature oligodendrocytes as well as the mature (sheath-forming) oligodendrocytes. Important in MS is the maintenance of myelin and remyelination following damage, thus the development of strategies to aid remyelination rely on manipulating oligodendrocytes in vitro. These in vitro models are also key to examine mechanisms of oligodendrocyte damage, such as apoptosis and oxidative stress. Importantly, primary rodent oligodendrocytes can be maintained for several weeks in vitro, and precursor oligodendrocytes can be differentiated to obtain the different maturation stages (Chen et al., 2007). A general method for isolating oligodendrocytes relies on their ability not to adhere to culture plates. By gentle shaking a flask of isolated CNS cells, the adherent microglia and astrocytes can be separated from the free-floating oligodendrocytes (McCarthy and de Vellis, 1980). Oligodendrocytes can also be obtained when glia progenitors are cultured in serum-free media (Raff et al., 1984) or differentiated from rodent stem cells (Chen et al., 2007; Czepiel et al., 2011), making these cells more accessible (De Vries and Boullerne, 2010; Seki et al., 2011). Primary human oligodendrocytes can be obtained post mortem while, more recently, oligodendrocyte progenitors have been obtained from differentiated human umbilical cord blood cells (Tracy et al., 2011). To obtain sufficient numbers of cells, oligodendrocyte cell lines have been created (Buntinx et al., 2003; Sundberg et al., 2010). Two commonly used examples are the OLN 93 and the Oli-neu cell lines. The OLN 93 cell line is derived from primary rat brain cultures and resembles bipolar O2A-progenitor cells. This cell line can be differentiated into immature oligodendrocytes when cultured under low serum conditions (Richter-Landsberg and Heinrich, 1996). In contrast, the Oli-neu is a mouse oligodendroglial precursor cell containing replication-defective retroviruses expressing the t-neu oncogene. Intriguingly, these cells interact with neurons and can therefore be used for culture systems to examine neuron–glia interactions (Jung et al., 1995). Thus, several sources of oligodendrocytes are available some of which are able to undergo differentiation to mature myelin forming oligodendrocytes. 6.4. Astrocytes Over a century ago, hyperplasia and hypertrophy of the astrocyte population was noted as a histopathological hallmark of MS and was hypothesised to play an important role in the development and course of this disease. Until today, however, the actual contribution of astrocytes to MS remains to be clarified. Astrocytes may play an active role during degeneration and demyelination by controlling local inflammation in the CNS, provoking damage of oligodendrocytes and axons, and glial scarring. They might also be beneficial by creating a permissive environment for remyelination and oligodendrocyte precursor migration, proliferation and differentiation (De Keyser et al., 2010; Kipp et al., 2011; Williams et al., 2007). For example, it has been demonstrated that glycosaminoglycan hyaluronan, synthesised by activated astrocytes accumulates in chronically demyelinated human MS lesions and inhibits oligodendrocyte progenitor cell (OPC) differentiation and consequently,

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remyelination (Back et al., 2005). On the other hand, astrocytes might contribute to regeneration and remyelination by secreting important growth factors, such as platelet-derived growth factor (PDGF), insulin-like growth factor 1 and basic fibroblast growth factor (Kipp et al., 2008, in press). All of these factors are known to promote remyelination and prevent oligodendrocyte apoptosis. The relevance of astrocytes in the pathology of MS has recently been reviewed (Moore et al., in press). Primary astrocyte cultures can be obtained from various starting materials including brain tissue from mice (Braun et al., 2009; Kipp et al., 2008), rats (McCarthy and de Vellis, 1980) and humans (De Groot et al., 1997). Usually, whole brain homogenates of newborn rats or mice are used. Protocols for the propagation of primary astrocyte cultures from adult animals have been reported as well (Codeluppi et al., 2011; Paluzzi et al., 2007). Furthermore, various stem cells can be differentiated towards an astrocyte lineage (Chen et al., 2007; Chojnacki and Weiss, 2008). Stimulated astrocytes are a relevant source of proinflammatory cytokines such as IL-1b, IL-6 or TNFa (Fernandes et al., 2011; Kipp et al., 2007, 2008), however conflicting results exist (Kim et al., 2011). In this context, microglia contamination of primary astrocyte cultures is relevant and can be avoided or at least diminished by treating the cultures with the specific microglia toxin L-leucine methyl ester (Kim et al., 2011). Recently, a non-expensive and fast staining method using the lysosomalaccumulating dye acridine orange was reported to detect microglia contamination in primary astrocyte cultures (Lovelace and Cahill, 2007).

oligodendrocyte damage. For example ablation of TNFR1 in mixed glia cultures completely prevents LPS-induced oligodendrocytes loss. To identify the role of individual cells oligodendrocytes were co-cultured with either wt or TNFR1-deficient mixed glia cultures. These studies showed that oligodendroglial TNFR1-expression is necessary for LPS-induced oligodendrocytes loss in mixed glia cultures (Kim et al., 2011). Finally, spatial separation in co-culture systems by special inserts or treatment of one monoculture with the conditioned medium from another one allows to determine, whether direct cell-cell contacts are a prerequisite for observed effects. 6.6. Spheroid and 3D CNS cultures To study demyelination and remyelination in vitro, CNS spheroids or 3D aggregate cultures have been created from different tissues and species, including the mouse telencephalon (Jackson et al., 2004), embryonic whole rat brain (Defaux et al., 2011; Vereyken et al., 2009) and human foetal brain (Hayes et al., 2000). Spheroids are generated by dissociating brain tissues to individual cells. When cultured under conditions of continual rotation, the cells gradually reassemble into clusters of cells. In these spheroids, or clusters, myelin is multilayered (Vereyken et al., 2009), resembling myelin in the CNS. Demyelination can be induced in rodent spheroid cultures using antibodies to MOG (Diemel et al., 2004; Jackson et al., 2004), LPC (Vereyken et al., 2009) or IFNg (Jackson et al., 2004). Since the spheroids are cocultures, systems can be established to examine interaction of different cell types with CNS tissues.

6.5. Co-cultures 6.7. Brain slice cultures Remyelination can be classified as four consecutive steps: (i) proliferation of oligodendrocyte progenitor cells, (ii) migration towards the demyelinated axons, (iii) differentiation and, finally (iv) interaction of the premature oligodendrocyte with the axon (i.e. axon wrapping) (Kipp et al., 2011; Shen et al., 2008). There are likely to be many factors contributing to the eventual failure of remyelination in MS and the essential processes of progenitor recruitment, differentiation, axon–oligodendrocyte interaction and myelination itself may all be adversely affected albeit to differing extents by a changing lesion environment and increasing levels of axonal compromise. As outlined above, primary oligodendrocytes differentiate at the morphological and biochemical level under proper culturing conditions. One major drawback of oligodendrocyte monoculture systems, however, is that interaction of the oligodendrocytes progenitor cell with the denuded axon is not modelled. Therefore, several co-culture approaches have been developed to be able to study this essential step in remyelination biology. The most frequently performed co-culture model applied is dorsal root ganglia cultured together with Schwann cells (Wan et al., 2010; Xiao et al., 2009) or Schwann cell-like cells derived from adult rat bone marrow (Shea et al., 2010). However, other neuronal cell types can be used such as primary cultured cortical neurons (Fex Svenningsen et al., 2003; Paez et al., 2005). Furthermore, other myelinating cell types than Schwann cells, such as oligodendrocyte cell lines (Paez et al., 2005) or primary oligodendrocytes (Wang et al., 2007) isolated following standard protocols (McCarthy and de Vellis, 1980) can be applied. Co-culture models are further useful to elaborate the impact of a certain cell type to oligodendrocyte injury. For example, OPCs are highly vulnerable to peroxynitrite but protected by astrocytes (Li et al., 2008). In these studies, mixed glia cultures are used which can be regarded as a co-culture system. Furthermore, cocultures from wild type and knock-out monoculture systems allow the study of the role of certain molecules in

A more complex in vitro model to study cell-cell interactions are brain slice cultures. Brain slice culture protocols have been reported for various species such as mice (Huang et al., 2011; Mi et al., 2009), rats (Birgbauer et al., 2004) and humans (Verwer et al., 2002). Slice cultures can be prepared from several brain regions including the cerebellum, forebrain (Mi et al., 2009) and hippocampus (Gimsa et al., 2000). A method of culturing ex vivo rat organotypic slices for electrophysiological recordings dates back to 1941 (Zhang et al., 2011), but myelination was first reported in longer term cerebellar slices in 1956 (Hild, 1956). Demyelination of these slices was achieved as early as 1959, by adding serum from animals with EAE (Bornstein and Appel, 1959). However, the technique was developed further to study myelination when immunohistochemical techniques were fully developed (Notterpek et al., 1993). In 2004, LPC was used to demyelinate rat cerebellar slices (Birgbauer et al., 2004). After the transient demyelinating insult the cultures recover with oligodendrocyte differentiation recapitulating a normal time course. LPC thus induces demyelination in an organotypic cerebellar slice culture system, providing a model system for studying myelination, demyelination, and remyelination in vitro. More recently this technique was used to investigate the action of exogenous molecules and drugs on the rate of CNS remyelination (Huang et al., 2011; Mi et al., 2009; Miron et al., 2010). Furthermore, the link between neurodegeneration and inflammation can be studied using appropriate brain slice culture models (Aktas et al., 2005; Dorr et al., 2005). 6.8. Blood–brain barrier models Approaches to examine the mechanisms of blood–brain barrier (BBB) damage in MS and the impact of e.g. therapies on infiltration of cells, has been investigated by developing in vitro systems to mimic the BBB. Cell culture systems that originate over


30 years ago originally used primary animal and human endothelial cells, and later immortalised brain endothelial cells. The awareness that the BBB is composed of astrocyte foot processes, basement membrane, and that the endothelial cells in the CNS have specific tight junctions aiding their function, has given rise to elaborate models (Wilhelm et al., 2011). For example, co-culture systems have been established using endothelial cells and astrocytes cultured on collagen-coated membranes in which transendothelial electrical resistance can be measured (citation). More recently, a dynamic humanised BBB model has been developed to study cell trafficking under flow conditions (Cucullo et al., 2011). While the latter appears to be the most ideal model, many BBB models are useful tools for drug discovery aimed at BBB repair and limiting cell influx into the CNS.

7. Perspectives and conclusions The choice of the experimental model ultimately depends on the research question and the availability of technical equipment for e.g. stereotactic injections. The clinical episodes of neurological disease in EAE models provide excellent opportunities to uncover immune mechanisms leading to both myelin damage and neuronal and axonal degeneration and dysfunction. For therapeutic studies, the chronic-relapsing models better reflect the human situation in MS, allowing strategies to inhibit established disease and promote neuroprotection. However, the emergence of infections in MS patients undergoing immunosuppressive therapies indicate a need for examining therapies also in a viral model, since EAE models have not and cannot predict infectious side effects of therapies. Some approaches examining mechanisms of tissue damage and therapies can be tested in human culture systems. The in vitro systems also have their limitations and several need refinement. In summary the perfect experimental model for MS is not available, partly because MS is a uniquely human disorder. Nevertheless, despite their limitations experimental in vitro and in vivo models continue to play an important role in neuroimmunology stimulating new ideas underlying MS and providing avenues for developing novel therapies.

Acknowledgments The authors thank MS Research; the multiple sclerosis society of the Netherlands, the multiple sclerosis of Great Britain and Northern Ireland, the Deutsche Forschungsgemeinschaft (DFG), Hertie foundation and the DANA foundation for financial support for studies involved in this review. Dr. Hans van Noort is gratefully acknowledged for his critical and constructive views on this manuscript. References Agholme L, Lindstrom T, Kagedal K, Marcusson J, Hallbeck M. An in vitro model for neuroscience: differentiation of SH-SY5Y cells into cells with morphological and biochemical characteristics of mature neurons. Journal of Alzheimer’s Disease 2010;20:1069–82. Aktas O, Smorodchenko A, Brocke S, Infante-Duarte C, Schulze Topphoff U, Vogt J, et al. Neuronal damage in autoimmune neuroinflammation mediated by the death ligand TRAIL. Neuron 2005;46:421–32. Al-Izki S, Pryce G, O’Neill JK, Butter C, Giovannoni G, Amor S. Practical guide to the induction of relapsing progressive experimental autoimmune encephalomyelitis in the Biozzi ABH mouse. Multiple Sclerosis and Related Disorders 2011;1:29–38. Amor S, Baker D, Groome N, Turk JL. Identification of a major encephalitogenic epitope of proteolipid protein (residues 56–70) for the induction of experimental allergic encephalomyelitis in Biozzi AB/H and nonobese diabetic mice. Journal of Immunology 1993;150:5666–72. Amor S, Groome N, Linington C, Morris MM, Dornmair K, Gardinier MV, et al. Identification of epitopes of myelin oligodendrocyte glycoprotein for the

25

induction of experimental allergic encephalomyelitis in SJL and Biozzi AB/H mice. Journal of Immunology 1994;153:4349–56. Amor S, Scallan MF, Morris MM, Dyson H, Fazakerley JK. Role of immune responses in protection and pathogenesis during Semliki Forest virus encephalitis. Journal of General Virology 1996a;77:281–91. Amor S, O’Neill JK, Morris MM, Smith RM, Wraith DC, Groome N, et al. Encephalitogenic epitopes of myelin basic protein, proteolipid protein, myelin oligodendrocyte glycoprotein for experimental allergic encephalomyelitis induction in Biozzi ABH (H-2Ag7) mice share an amino acid motif. Journal of Immunology 1996b;156:3000–8. Back SA, Tuohy TM, Chen H, Wallingford N, Craig A, Struve J, et al. Hyaluronan accumulates in demyelinated lesions and inhibits oligodendrocyte progenitor maturation. Nature Medicine 2005;11:966–72. Bajramovic JJ, Brok HP, Ouwerling B, Jagessar SA, van Straalen L, Kondova I, et al. Oligodendrocyte-specific protein is encephalitogenic in rhesus macaques and induces specific demyelination of the optic nerve. European Journal of Immunology 2008;38:1452–64. Baker D, Gerritsen W, Rundle J, Amor S. Critical appraisal of animal models of multiple sclerosis. Multiple Sclerosis 2011;7(6):647–57. Baker D, O’Neill JK, Gschmeissner SE, Wilcox CE, Butter C, Turk JL. Induction of chronic relapsing experimental allergic encephalomyelitis in Biozzi mice. Journal of Neuroimmunology 1990;28:261–70. Baxter AG. The origin and application of experimental autoimmune encephalomyelitis. Nature Reviews Immunology 2007;7:904–12. Benavides J, Garcia-Pariente C, Fuertes M, Ferreras MC, Garcia-Marin JF, Juste RA, et al. Maedi-visna: the meningoencephalitis in naturally occurring cases. Journal of Comparative Pathology 2009;140:1–11. Bertrand SJ, Aksenova MV, Aksenov MY, Mactutus CF, Booze RM. Endogenous amyloidogenesis in long-term rat hippocampal cell cultures. BMC Neuroscience 2011;12:38. Bettelli E, Pagany M, Weiner HL, Linington C, Sobel RA, Kuchroo VK. Myelin oligodendrocyte glycoprotein-specific T cell receptor transgenic mice develop spontaneous autoimmune optic neuritis. Journal of Experimental Medicine 2003;197:1073–81. Birgbauer E, Rao TS, Webb M. Lysolecithin induces demyelination in vitro in a cerebellar slice culture system. Journal of Neuroscience Research 2004;78:157–66. Blakemore WF. Lesions in the cat spinal cord following local injections of 6-aminonicotinamide. Research in Veterinary Science 1978;24:390–1. Blakemore WF, Chari DM, Gilson JM, Crang AJ. Modelling large areas of demyelination in the rat reveals the potential and possible limitations of transplanted glial cells for remyelination in the CNS. Glia 2002;38:155–68. Blakemore WF, Franklin RJ. Remyelination in experimental models of toxininduced demyelination. Current Topics in Microbiology and Immunology 2008;318:193–212. Bornstein MB, Appel SH. Demyelination in cultures of rat cerebellum produced by experimental allergic encephalomyelitic serum. Transactions of the American Neurological Association 1959;84:165–6. Braun A, Dang J, Johann S, Beyer C, Kipp M. Selective regulation of growth factor expression in cultured cortical astrocytes by neuro-pathological toxins. Neurochemistry International 2009;55:610–8. Brewer GJ, Espinosa J, McIlhaney MP, Pencek TP, Kesslak JP, Cotman C, et al. Culture and regeneration of human neurons after brain surgery. Journal of Neurosciences Methods 2001;107:15–23. Buntinx M, Vanderlocht J, Hellings N, Vandenabeele F, Lambrichts I, Raus J, et al. Characterization of three human oligodendroglial cell lines as a model to study oligodendrocyte injury: morphology and oligodendrocyte-specific gene expression. Journal of Neurocytology 2003;32:25–38. Cano-Abad MF, Herrera-Peco I, Sola RG, Pastor J, Garcia-Navarrete E, Moro RC, et al. New insights on culture and calcium signalling in neurons and astrocytes from epileptic patients. International Journal of Devlopmental Neuroscience 2011;29:121–9. Carlton WW. Spongiform encephalopathy induced in rats and guinea pigs by cuprizone. Experimental and Molecular Pathology 1969;10:274–87. Carroll WM, Jennings A, Mastaglia FL. Experimental demyelinating optic neuropathy: a model for combined morphological and electrophysiological studies. Clinical and Experimental Neurology 1983;19:17–28. Castegna A, Palmieri L, Spera I, Porcelli V, Palmieri F, Fabis-Pedrini MJ, et al. Oxidative stress and reduced glutamine synthetase activity in the absence of inflammation in the cortex of mice with experimental allergic encephalomyelitis. Neuroscience 2011;185:97–105. Chen K, Hughes SM, Connor B. Neural progenitor cells derived from the adult rat subventricular zone: characterization and transplantation. Cell Transplant 2007;6:799–810. Chojnacki A, Weiss S. Production of neurons, astrocytes and oligodendrocytes from mammalian CNS stem cells. Nature Protocols 2008;3:35–40. Codeluppi S, Gregory EN, Kjell J, Wigerblad G, Olson L, Svensson CI. Influence of rat substrain and growth conditions on the characteristics of primary cultures of adult rat spinal cord astrocytes. Journal of Neurosciences Methods 2011;197: 118–27. Copray S, Balasubramaniyan V, Levenga J, de Bruijn J, Liem R, Boddeke E. Olig2 overexpression induces the in vitro differentiation of neural stem cells into mature oligodendrocytes. Stem Cells 2006;24:1001–10. Cucullo L, Marchi N, Hossain M, Janigro D. A dynamic in vitro BBB model for the study of immune cell trafficking into the central nervous system. Journal of Cerebral Blood Flow and Metabolism 2011;31:767–77.

26


Czepiel M, Balasubramaniyan V, Schaafsma W, Stancic M, Mikkers H, Huisman C, et al. S. Differentiation of induced pluripotent stem cells into functional oligodendrocytes. Glia 2011;59:882–92. Dandekar AA, Wu GF, Pewe L, Perlman S. Axonal damage is T cell mediated and occurs concomitantly with demyelination in mice infected with a neurotropic coronavirus. Journal of Virology 2001;75:6115–20. Das Sarma J, Kenyon LC, Hingley ST, Shindler KS. Mechanisms of primary axonal damage in a viral model of multiple sclerosis. Journal of Neuroscience 2009;29:10272–80. De Groot CJ, Langeveld CH, Jongenelen CA, Montagne L, Van Der Valk P, Dijkstra CD. Establishment of human adult astrocyte cultures derived from postmortem multiple sclerosis and control brain and spinal cord regions: immunophenotypical and functional characterization. Journal of Neuroscience Research 1997;49:342–54. De Groot CJ, Hulshof S, Hoozemans JJ, Veerhuis R. Establishment of microglial cell cultures derived from postmortem human adult brain tissue: immunophenotypical and functional characterization. Microscopy Research and Technique 2001;54:34–9. De Haas AH, Boddeke HW, Brouwer N, Biber K. Optimized isolation enables ex vivo analysis of microglia from various central nervous system regions. Glia 2007;55:1374–84. De Keyser J, Laureys G, Demol F, Wilczak N, Mostert J, Clinckers R. Astrocytes as potential targets to suppress inflammatory demyelinating lesions in multiple sclerosis. Neurochemistry International 2010;57:446–50. De Vries GH, Boullerne AI. Glial cell lines: an overview. Neurochemical Research 2010;35:1978–2000. Defaux A, Zurich MG, Honegger P, Monnet-Tschudi F. Minocycline promotes remyelination in aggregating rat brain cell cultures after interferon-gamma plus lipopolysaccharide-induced demyelination. Neuroscience 2011;187:84–92. Degaonkar MN, Jayasundar R, Jagannathan NR. Sequential diffusion-weighted magnetic resonance imaging study of lysophosphatidyl choline-induced experimental demyelinating lesion: an animal model of multiple sclerosis. Journal of Magnetic Resonance Imaging 2002;16:153–9. Derfuss T, Parikh K, Velhin S, Braun M, Mathey E, Krumbholz M, et al. Contactin-2/ TAG-1-directed autoimmunity is identified in multiple sclerosis patients and mediates gray matter pathology in animals. Proceedings of the National Academy of Sciences USA 2009;106:8302–7. Diemel LT, Wolswijk G, Jackson SJ, Cuzner ML. Remyelination of cytokine- or antibody-demyelinated CNS aggregate cultures is inhibited by macrophage supplementation. Glia 2004;45:278–86. Ditamo Y, Degano AL, Maccio DR, Pistoresi-Palencia MC, Roth GA. Age-related changes in the development of experimental autoimmune encephalomyelitis. Immunology and Cell Biology 2005;83:75–82. Dorr J, Roth K, Zurbuchen U, Deisz R, Bechmann I, Lehmann TN, et al. Tumornecrosis-factor-related apoptosis-inducing-ligand (TRAIL)-mediated death of neurons in living human brain tissue is inhibited by flupirtine-maleate. Journal of Neuroimmunology 2005;67:204–9. Dromard C, Guillon H, Rigau V, Ripoll C, Sabourin JC, Perrin FE, et al. Adult human spinal cord harbors neural precursor cells that generate neurons and glial cells in vitro. Journal of Neuroscience Research 2008;86:1916–26. Ellmerich S, Mycko M, Takacs K, Waldner H, Wahid FN, Boyton RJ, et al. High incidence of spontaneous disease in an HLA-DR15 and TCR transgenic multiple sclerosis model. Journal of Immunology 2005;174:1938–46. Emerson MR, Biswas S, LeVine SM. Cuprizone and piperonyl butoxide, proposed inhibitors of T-cell function, attenuate experimental allergic encephalomyelitis in SJL mice. Journal of Neuroimmunology 2001;119:205–13. Evron S, Brenner T, Abramsky O. Suppressive effect of pregnancy on the development of experimental allergic encephalomyelitis in rabbits. American Journal of Reproductive Immunology 1984;5:109–13. Fazakerley JK, Amor S, Webb HE. Reconstitution of Semliki forest virus infected mice, induces immune mediated pathological changes in the CNS. Clinical and Experimental Immunology 1983;52:115–20. Fazakerley JK, Walker R. Virus demyelination. Journal for Neurovirology 2003;9:148–64. Felts PA, Woolston AM, Fernando HB, Asquith S, Gregson NA, Mizzi OJ, et al. Inflammation and primary demyelination induced by the intraspinal injection of lipopolysaccharide. Brain 2005;128:1649–66. Fernandes A, Barateiro A, Falcao AS, Silva SL, Vaz AR, Brito MA, et al. Astrocyte reactivity to unconjugated bilirubin requires TNF-alpha and IL-1beta receptor signaling pathways. Glia 2011;59:14–25. Fex Svenningsen A, Shan WS, Colman DR, Pedraza L. Rapid method for culturing embryonic neuron–glial cell cocultures. Journal of Neuroscience Research 2003;72:565–73. Ford AL, Goodsall AL, Hickey WF, Sedgwick JD. Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4 þ T cells compared. Journal of Immunology 1995;154:4309–21. Forsby A, Bal-Price AK, Camins A, Coecke S, Fabre N, Gustafsson H, et al. Neuronal in vitro models for the estimation of acute systemic toxicity. Toxicology In Vitro 2009;23:1564–9. Fragkoudis R, Tamberg N, Siu R, Kiiver K, Kohl A, Merits A, et al. Neurons and oligodendrocytes in the mouse brain differ in their ability to replicate Semliki Forest virus. Journal for Neurovirology 2009;15:57–70. Franklin RJ, Crang AJ, Blakemore WF. The reconstruction of an astrocytic environment in glia-deficient areas of white matter. Journal of Neurocytology 1993;22:382–96.

Frei K, Bodmer S, Schwerdel C, Fontana A. Astrocyte-derived interleukin 3 as a growth factor for microglia cells and peritoneal macrophages. Journal of Immunology 1986;137:3521–7. Gambi D, Di Cesare N, Di Trapani G, Macchi G, Sbriccoli A. Experimental allergic encephalomyelitis in guinea pig: variability of response to intradermal emulsion injection. Italian Journal of Neurological Science 1989;10:33–41. Ganter S, Northoff H, Mannel D, Gebicke-Harter PJ. Growth control of cultured microglia. Journal of Neuroscience Research 1992;33:218–30. Genain CP, Hauser SL. Experimental allergic encephalomyelitis in the New World monkey Callithrix jacchus. Immunology Review 2001;183:159–72. Gibbons HM, Dragunow M. Adult human brain cell culture for neuroscience research. International Journal for Biochemistry and Cell Biology 2010;42: 844–56. Gimsa U, Peter SV, Lehmann K, Bechmann I, Nitsch R. Axonal damage induced by invading T cells in organotypic central nervous system tissue in vitro: involvement of microglial cells. Brain Pathology 2000;10:365–77. Giulian D, Baker TJ. Characterization of ameboid microglia isolated from developing mammalian brain. Journal of Neuroscience 1986;6:2163–78. Glasgow GM, McGee MM, Sheahan BJ, Atkins GJ. Death mechanisms in cultured cells infected by Semliki Forest virus. Journal of General Virology 1997;78: 1559–63. Goudarzvand M, Javan M, Mirnajafi-Zadeh J, Mozafari S, Tiraihi T. Vitamins E and D3 attenuate demyelination and potentiate remyelination processes of hippocampal formation of rats following local injection of ethidium bromide. Cell and Molecular Neurobiology 2010;30:289–99. Gregson NA. Lysolipids and membrane damage: lysolecithin and its interaction with myelin. Biochemistry Society Transaction 1989;17:280–3. Guazzo EP. A technique for producing demyelination of the rat optic nerves. Journal of Clinical Neuroscience 2005;12:54–8. Hall SM. Some aspects of remyelination after demyelination produced by the intraneural injection of lysophosphatidyl choline. Journal Cell Science 1973;13: 461–77. Hampton DW, Anderson J, Pryce G, Irvine KA, Giovannoni G, Fawcett JW, et al. An experimental model of secondary progressive multiple sclerosis that shows regional variation in gliosis, remyelination, axonal and neuronal loss. Journal of Neuroimmunology 2008;201-202:200–11. Hao C, Richardson A, Fedoroff S. Macrophage-like cells originate from neuroepithelium in culture: characterization and properties of the macrophage-like cells. International Journal of Devlopmental Neuroscience 1991;9:1–14. Hayes GM, Fox RM, Cuzner ML, Griffin GE. Human rotation-mediated fetal mixed brain cell aggregate culture: characterization and N-methyl-D-aspartate toxicity. Neuroscience Letters 2000;287:146–50. Heijmans N, Smith PA, Morris-Downes MM, Pryce G, Baker D, Donaldson AV, et al. Encephalitogenic and tolerogenic potential of altered peptide ligands of MOG and PLP in Biozzi ABH mice. Journal of Neuroimmunology 2005;167:23–33. Hild W. Myelin formation in central nervous system tissue cultures. Verhandlungen der Anatomischen Gesellschaft 1956;53:315–7. Howe CL, Adelson JD, Rodriguez M. Absence of perforin expression confers axonal protection despite demyelination. Neurobiology of Disease 2007;25:354–9. Huang JH, Zager EL, Zhang J, Groff RF, Pfister BJ, Cohen AS, et al. Harvested human neurons engineered as live nervous tissue constructs: implications for transplantation. Laboratory investigation. Journal of Neurosurgery 2008;108: 343–7. Huang JK, Jarjour AA, Nait Oumesmar B, Kerninon C, Williams A, Krezel W, et al. Retinoid X receptor gamma signaling accelerates CNS remyelination. Nature Neuroscience 2011;14:45–53. Huizinga R, Heijmans N, Schubert P, Gschmeissner S, t Hart BA, Herrmann H, et al. Immunization with neurofilament light protein induces spastic paresis and axonal degeneration in Biozzi ABH mice. Journal of Neuropathology and Experimental Neurology 2007;66:295–304. Huizinga R, Gerritsen W, Heijmans N, Amor S. Axonal loss and gray matter pathology as a direct result of autoimmunity to neurofilaments. Neurobiology of Disease 2008;32:461–70. Jablonska B, Aguirre A, Raymond M, Szabo G, Kitabatake Y, Sailor KA, et al. Chordin-induced lineage plasticity of adult SVZ neuroblasts after demyelination. Nature Neuroscience 2010;13:541–50. Jackson SJ, Baker D, Cuzner ML, Diemel LT. Cannabinoid-mediated neuroprotection following interferon-gamma treatment in a three-dimensional mouse brain aggregate cell culture. European Journal of Neuroscience 2004;20:2267–75. Jagessar SA, Smith PA, Blezer E, Delarasse C, Pham-Dinh D, Laman JD, et al. Autoimmunity against myelin oligodendrocyte glycoprotein is dispensable for the initiation although essential for the progression of chronic encephalomyelitis in common marmosets. Journal of Neuropathology and Experimental Neurology 2008;67:326–40. Jha S, Srivastava SY, Brickey WJ, Iocca H, Toews A, Morrison JP, et al. The inflammasome sensor, NLRP3, regulates CNS inflammation and demyelination via caspase-1 and interleukin-18. Journal of Neuroscience 2010;30:15811–20. Johnson TA, Jirik FR, Fournier S. Exploring the roles of CD8( þ ) T lymphocytes in the pathogenesis of autoimmune demyelination. Seminars in Immunopathology 2010;32:197–209. Jung M, Kramer E, Grzenkowski M, Tang K, Blakemore W, Aguzzi A, et al. Lines of murine oligodendroglial precursor cells immortalized by an activated neu tyrosine kinase show distinct degrees of interaction with axons in vitro and in vivo. European Journal of Neuroscience 1995;7:1245–65.


Kabat EA, Wolf A, Bezer AE. Rapid production of acute disseminated encephalomyelitis in rhesus monkeys by injection of brain tissue with adjuvants. Science 1946;104:362. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biology 2010;8:e1000412. Kim S, Steelman AJ, Koito H, Li J. Astrocytes promote TNF-mediated toxicity to oligodendrocyte precursors. Journal of Neurochemistry 2011;116:53–66. Kipp M, Karakaya S, Johann S, Kampmann E, Mey J, Beyer C. Oestrogen and progesterone reduce lipopolysaccharide-induced expression of tumour necrosis factor-alpha and interleukin-18 in midbrain astrocytes. Journal of Neuroendocrinology 2007;19:819–22. Kipp M, Norkute A, Johann S, Lorenz L, Braun A, Hieble A, et al. Brain-regionspecific astroglial responses in vitro after LPS exposure. Journal of Molecular Neuroscience 2008;35:235–43. Kipp M, Clarner T, Dang J, Copray S, Beyer C. The cuprizone animal model: new insights into an old story. Acta Neuropathology 2009;118:723–36. Kipp M, Gingele S, Pott F, Clarner T, van der Valk P, Denecke B, et al. BLBPexpression in astrocytes during experimental demyelination and in human multiple sclerosis lesions. Brain Behaviour and Immunity, 2011. Kitazawa A, Shimizu N. Differentiation of mouse embryonic stem cells into neurons using conditioned medium of dorsal root ganglia. Journal of Bioscience and Bioengineering 2005;100:94–9. Kreutzberg GW. Microglia: a sensor for pathological events in the CNS. Trends in Neuroscience 1996;19:312–8. Krishnamoorthy G, Lassmann H, Wekerle H, Holz A. Spontaneous opticospinal encephalomyelitis in a double-transgenic mouse model of autoimmune T cell/ B cell cooperation. Journal of Clinical Investigation 2006;116:2385–422. Kume N, Cybulsky MI, Gimbrone Jr. MA. Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells. Journal of Clinical Investigation 1992;90:1138–44. Lehnardt S, Lachance C, Patrizi S, Lefebvre S, Follett PL, Jensen FE, et al. The tolllike receptor TLR4 is necessary for lipopolysaccharide-induced oligodendrocyte injury in the CNS. Journal of Neuroscience 2002;22:2478–86. Leone DP, Genoud S, Atanasoski S, Grausenburger R, Berger P, Metzger D, et al. Tamoxifen-inducible glia-specific Cre mice for somatic mutagenesis in oligodendrocytes and Schwann cells. Molecular and Cellular Neuroscience 2003;22:430–40. Levine S, Sowinski R. Hyperacute allergic encephalomyelitis. A localized form produced by passive transfer and pertussis vaccine. American Journal of Pathology 1973;73:247–60. Li J, Ramenaden ER, Peng J, Koito H, Volpe JJ, Rosenberg PA. Tumor necrosis factor alpha mediates lipopolysaccharide-induced microglial toxicity to developing oligodendrocytes when astrocytes are present. Journal of Neuroscience 2008;28:5321–30. Liebert UG, Hashim GA, ter Meulen V. Characterization of measles virus-induced cellular autoimmune reactions against myelin basic protein in Lewis rats. Journal of Neuroimmunology 1990;29:139–47. Lorentzen JC, Issazadeh S, Storch M, Mustafa MI, Lassman H, Linington C, et al. Protracted, relapsing and demyelinating experimental autoimmune encephalomyelitis in DA rats immunized with syngeneic spinal cord and incomplete Freund’s adjuvant. Journal of Neuroimmunology 1995;63:193–205. Lorenz L, Dang J, Misiak M, Tameh Abolfazl A, Beyer C, Kipp M. Combined 17betaoestradiol and progesterone treatment prevents neuronal cell injury in cortical but not midbrain neurones or neuroblastoma cells. Journal of Neuroendocrinology 2009;21:841–9. Lovelace MD, Cahill DM. A rapid cell counting method utilising acridine orange as a novel discriminating marker for both cultured astrocytes and microglia. Journal of Neurosciences Methods 2007;65:223–9. MacKenzie-Graham A, Tinsley MR, Shah KP, Aguilar C, Strickland LV, Boline J, et al. Cerebellar cortical atrophy in experimental autoimmune encephalomyelitis. Neuroimage 2006;32:1016–23. Mangiardi M, Crawford DK, Xia X, Du S, Simon-Freeman R, Voskuhl RR, et al. An animal model of cortical and callosal pathology in multiple sclerosis. Brain Pathology 2011;21:263–78. Matejuk A, Hopke C, Vandenbark AA, Hurn PD, Offner H. Middle-age male mice have increased severity of experimental autoimmune encephalomyelitis and are unresponsive to testosterone therapy. Journal of Immunology 2005;174:2387–95. Mathey EK, Derfuss T, Storch MK, Williams KR, Hales K, Woolley DR, et al. Neurofascin as a novel target for autoantibody-mediated axonal injury. Journal of Experimental Medicine 2007;204:2363–72. Matsushima GK, Morell P. The neurotoxicant, cuprizone, as a model to study demyelination and remyelination in the central nervous system. Brain Pathology 2001;11:107–16. Matyszak MK, Perry VH. Demyelination in the central nervous system following a delayed-type hypersensitivity response to bacillus Calmette-Guerin. Neuroscience 1995;64:967–77. McCarthy KD, de Vellis J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. Journal of Cell Biology 1980;85:890–902. McMahon EJ, Suzuki K, Matsushima GK. Peripheral macrophage recruitment in cuprizone-induced CNS demyelination despite an intact blood–brain barrier. Journal of Neuroimmunology 2002;30:32–45. Mendel I, Kerlero de Rosbo N, Ben-Nun A. A myelin oligodendrocyte glycoprotein peptide induces typical chronic experimental autoimmune encephalomyelitis

27

in H-2b mice: fine specificity and T cell receptor V beta expression of encephalitogenic T cells. European Journal of Immunology 1995;25:1951–9. Merkler D, Ernsting T, Kerschensteiner M, Bruck W, Stadelmann C. A new focal EAE model of cortical demyelination: multiple sclerosis-like lesions with rapid resolution of inflammation and extensive remyelination. Brain 2006;129: 1972–83. Mi S, Miller RH, Tang W, Lee X, Hu B, Wu W, et al. Promotion of central nervous system remyelination by induced differentiation of oligodendrocyte precursor cells. Annals of Neurology 2009;65:304–15. Mildner A, Schmidt H, Nitsche M, Merkler D, Hanisch UK, Mack M, et al. Microglia in the adult brain arise from Ly-6ChiCCR2 þ monocytes only under defined host conditions. Nature Neuroscience 2007;10:1544–53. Miron VE, Ludwin SK, Darlington PJ, Jarjour AA, Soliven B, Kennedy TE, et al. Fingolimod (FTY720) enhances remyelination following demyelination of organotypic cerebellar slices. American Journal of Pathology 2010;176: 2682–94. Mokhtarian F, Huan CM, Roman C, Raine CS. Semliki Forest virus-induced demyelination and remyelination—involvement of B cells and anti-myelin antibodies. Journal of Neuroimmunology 2003;137:19–31. Morris-Downes MM, Smith PA, Rundle JL, Piddlesden SJ, Baker D, Pham-Dinh D, et al. Pathological and regulatory effects of anti-myelin antibodies in experimental allergic encephalomyelitis in mice. Journal of Neuroimmunology 2002a;125:114–24. Morris-Downes MM, McCormack K, Baker D, Sivaprasad D, Natkunarajah J, Amor S. Encephalitogenic and immunogenic potential of myelin-associated glycoprotein (MAG), oligodendrocyte-specific glycoprotein (OSP) and 20 ,30 -cyclic nucleotide 30 -phosphodiesterase (CNPase) in ABH and SJL mice. Journal of Neuroimmunology 2002b;122:20–33. Moore CS, Abdullah SL, Brown A, Arulpragasam A, Crocker SJ. How factors secreted from astrocytes impact myelin repair. Journal of Neuroscience Research, in press. Mothe AJ, Tator CH. Transplanted neural stem/progenitor cells generate myelinating oligodendrocytes and Schwann cells in spinal cord demyelination and dysmyelination. Experimental Neurology 2008;213:176–90. Nagai A, Nakagawa E, Hatori K, Choi HB, McLarnon JG, Lee MA, et al. Generation and characterization of immortalized human microglial cell lines: expression of cytokines and chemokines. Neurobiology of Disease 2001;8:1057–68. Napoli I, Kierdorf K, Neumann H. Microglial precursors derived from mouse embryonic stem cells. Glia 2009;57:1660–71. Napoli I, Neumann H. Protective effects of microglia in multiple sclerosis. Experimental Neurology 2010;225:24–8. Neumann H, Kotter MR, Franklin RJ. Debris clearance by microglia: an essential link between degeneration and regeneration. Brain 2009;132:288–95. Nikic I, Merkler D, Sorbara C, Brinkoetter M, Kreutzfeldt M, Bareyre FM, et al. A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis. Nature Medicine 2011;17:495–9. Norkute A, Hieble A, Braun A, Johann S, Clarner T, Baumgartner W, et al. Cuprizone treatment induces demyelination and astrocytosis in the mouse hippocampus. Journal of Neuroscience Research 2009;87:1343–55. Notterpek LM, Bullock PN, Malek-Hedayat S, Fisher R, Rome LH. Myelination in cerebellar slice cultures: development of a system amenable to biochemical analysis. Journal of Neuroscience Research 1993;36:621–34. Ousman SS, David S. Lysophosphatidylcholine induces rapid recruitment and activation of macrophages in the adult mouse spinal cord. Glia 2000;30: 92–104. Ousman SS, Tomooka BH, van Noort JM, Wawrousek EF, O’Connor KC, Hafler DA, et al. Protective and therapeutic role for alpha B-crystallin in autoimmune demyelination. Nature 2007;448:474–9. Paez PM, Garcia CI, Campagnoni AT, Soto EF, Pasquini JM. Overexpression of human transferrin in two oligodendroglial cell lines enhances their differentiation. Glia 2005;52:1–15. Paluzzi S, Alloisio S, Zappettini S, Milanese M, Raiteri L, Nobile M, et al. Adult astroglia is competent for Naþ /Ca2þ exchanger-operated exocytotic glutamate release triggered by mild depolarization. Journal of Neurochemistry 2007;103:1196–207. Penderis J, Shields SA, Franklin RJ. Impaired remyelination and depletion of oligodendrocyte progenitors does not occur following repeated episodes of focal demyelination in the rat central nervous system. Brain 2003;126: 1382–91. Perry VH, Nicoll JA, Holmes C. Microglia in neurodegenerative disease. Nature Reviews Neurology 2010;6:193–201. Pettinelli CB, McFarlin DE. Adoptive transfer of experimental allergic encephalomyelitis in SJL/J mice after in vitro activation of lymph node cells by myelin basic protein: requirement for Lyt 1 þ 2 T lymphocytes. Journal of Immunology 1981;127:1420–3. Pleasure SJ, Page C, Lee VM. Pure, postmitotic, polarized human neurons derived from NTera 2 cells provide a system for expressing exogenous proteins in terminally differentiated neurons. Journal of Neuroscience 1992;12:1802–15. Pohl HB, Porcheri C, Mueggler T, Bachmann LC, Martino G, Riethmacher D, et al. Genetically induced adult oligodendrocyte cell death is associated with poor myelin clearance, reduced remyelination, and axonal damage. Journal of Neuroscience 2011;31:1069–80. Pomeroy IM, Jordan EK, Frank JA, Matthews PM, Esiri MM. Diffuse cortical atrophy in a marmoset model of multiple sclerosis. Neuroscience Letters 2008;437: 121–4.

28


Pringproa K, Rohn K, Kummerfeld M, Wewetzer K, Baumgartner W. Theiler’s murine encephalomyelitis virus preferentially infects immature stages of the murine oligodendrocyte precursor cell line BO-1 and blocks oligodendrocytic differentiation in vitro. Brain Research 2010;1327:24–37. Pryce G, O’Neill JK, Croxford JL, Amor S, Hankey DJ, East E, et al. Autoimmune tolerance eliminates relapses but fails to halt progression in a model of multiple sclerosis. Journal of Neuroimmunology 2005;165:41–52. Quinn MT, Parthasarathy S, Steinberg D. Lysophosphatidylcholine: a chemotactic factor for human monocytes and its potential role in atherogenesis. Proceedings of the National Academy of Sciences USA 1988;85:2805–9. Raasch J, Zeller N, van Loo G, Merkler D, Mildner A, Erny D, et al. kappaB kinase 2 determines oligodendrocyte loss by non-cell-autonomous activation of NFkappaB in the central nervous system. Brain 2011;134:1184–98. Raff MC, Williams BP, Miller RH. The in vitro differentiation of a bipotential glial progenitor cell. EMBO Journal 1984;3:1857–64. Raine CS, Traugott U, Stone SH. Chronic relapsing experimental allergic encephalomyelitis: CNS plaque development in unsuppressed and suppressed animals. Acta Neuropathologica 1978;43:43–53. Remington LT, Babcock AA, Zehntner SP, Owens T. Microglial recruitment, activation, and proliferation in response to primary demyelination. American Journal of Pathology 2007;170:1713–24. Reynolds R, di Bello IC, Meeson A, Piddlesden S. Comparison of a Chemically Mediated and an Immunologically Mediated Demyelinating Lesion Model. Methods 1996;10:440–52. Richard JF, Roy M, Audoy-Re´mus J, Tremblay P, Vallie res L. Crawling phagocytes recruited in the brain vasculature after pertussis toxin exposure through IL6, ICAM1 and ITGaM. Brain Pathology 2011, March 22. doi:10.1111/j.1750-3639. 2011.00490.x. Richter-Landsberg C, Heinrich M. OLN-93: a new permanent oligodendroglia cell line derived from primary rat brain glial cultures. Journal of Neuroscience Research 1996;45:161–73. Rivers TM, Schwentker FF. Encephalomyelitis accompanied by myelin destruction experimentally produced in monkeys. Journal of Experimental Medicine 1935;61:689–702. Rojiani AM, Prineas JW, Cho ES. Electrolyte-induced demyelination in rats. 1. Role of the blood–brain barrier and edema. Acta Neuropathologica 1994;88: 287–92. Rosenbluth J, Schiff R, Liang WL, Dou W. Antibody-mediated CNS demyelination II. Focal spinal cord lesions induced by implantation of an IgM antisulfatidesecreting hybridoma. Journal of Neurocytology 2003;32:265–76. Rosenbluth J, Schiff R. Spinal cord dysmyelination caused by an antiproteolipid protein IgM antibody: implications for the mechanism of central nervous system myelin formation. Journal of Neuroscience Research 2009;87:956–63. Salli U, Reddy AP, Salli N, Lu NZ, Kuo HC, Pau FK, et al. Serotonin neurons derived from rhesus monkey embryonic stem cells: similarities to CNS serotonin neurons. Experimental Neurology 2004;188:351–64. Sasaki M, Lankford KL, Brown RJ, Ruddle NH, Kocsis JD. Focal experimental autoimmune encephalomyelitis in the Lewis rat induced by immunization with myelin oligodendrocyte glycoprotein and intraspinal injection of vascular endothelial growth factor. Glia 2010;58:1523–31. Scheikl T, Pignolet B, Mars LT, Liblau RS. Transgenic mouse models of multiple sclerosis. Cellular and Molecular Life Sciences 2010;67:4011–34. Seki Y, Suzuki SO, Masui K, Harada S, Nakamura S, Kanba S, et al. A simple and high-yield method for preparation of rat microglial cultures utilizing Aclar plastic film. Neuropathology 2011;31:215–22. Serres S, Anthony DC, Jiang Y, Broom KA, Campbell SJ, Tyler DJ, et al. Systemic inflammatory response reactivates immune-mediated lesions in rat brain. Journal of Neuroscience 2009;29:4820–8. Shea GK, Tsui AY, Chan YS, Shum DK. Bone marrow-derived Schwann cells achieve fate commitment—a prerequisite for remyelination therapy. Experimental Neurology 2010;224:448–58. Shen S, Sandoval J, Swiss VA, Li J, Dupree J, Franklin RJ, et al. Age-dependent epigenetic control of differentiation inhibitors is critical for remyelination efficiency. Nature Neuroscience 2008;11:1024–34. Shindler KS, Kenyon LC, Dutt M, Hingley ST, Das Sarma J. Experimental optic neuritis induced by a demyelinating strain of mouse hepatitis virus. Journal of Virology 2008;82:8882–6. Skuljec J, Sun H, Pul R, Benardais K, Ragancokova D, Moharregh-Khiabani D, et al. CCL5 induces a pro-inflammatory profile in microglia in vitro. Cellular Immunology, in press. Smith KJ, Bostock H, Hall SM. Saltatory conduction precedes remyelination in axons demyelinated with lysophosphatidyl choline. Journal of the Neurological Sciences 1982;54:13–31. Smith PA, Heijmans N, Ouwerling B, Breij EC, Evans N, van Noort JM, et al. Native myelin oligodendrocyte glycoprotein promotes severe chronic neurological disease and demyelination in Biozzi ABH mice. European Journal of Immunology 2005;35:1311–9. Smithburn KC, Haddow AJ, Mahaffy AF. A neurotropic virus isolated from Aedes mosquitoes caught in the Semliki forest. American Journal of Tropical Medicine and Hygiene 1946;26:189–208. Soilu-Hanninen M, Roytta M, Salmi A. Salonen, RTherapy with antibody against leukocyte integrin VLA-4 (CD49d) is effective and safe in virus-facilitated

experimental allergic encephalomyelitis. Journal of Neuroimmunology 1997;72: 95–105. Sundberg M, Skottman H, Suuronen R, Narkilahti S. Production and isolation of NG2 þ oligodendrocyte precursors from human embryonic stem cells in defined serum-free medium. Stem Cell Research 2010;5:91–103. Swanborg RH. Experimental autoimmune encephalomyelitis in the rat: lessons in T-cell immunology and autoreactivity. Immunological Reviews 2001;184: 129–35. Talbott JF, Cao Q, Enzmann GU, Benton RL, Achim V, Cheng XX, et al. Schwann celllike differentiation by adult oligodendrocyte precursor cells following engraftment into the demyelinated spinal cord is BMP-dependent. Glia 2006;54: 147–59. Taylor LC, Gilmore W, Matsushima GK. SJL mice exposed to cuprizone intoxication reveal strain and gender pattern differences in demyelination. Brain Pathology 2009;19:467–79. Theiler M. Spontaneous encephalomyelitis of mice—a new virus disease. Science 1934;80:122. Tourdias T, Hiba B, Raffard G, Biran M, Nishiguchi T, Aussudre J, et al. Adapted focal experimental autoimmune encephalomyelitis to allow MRI exploration of multiple sclerosis features. Experimental Neurology, in press. Tracy ET, Zhang CY, Gentry T, Shoulars KW, Kurtzberg J. Isolation and expansion of oligodendrocyte progenitor cells from cryopreserved human umbilical cord blood. Cytotherapy, 2011. Triarhou LC, Herndon RM. The effect of dexamethasone on L-alpha-lysophosphatidyl choline (lysolecithin)-induced demyelination of the rat spinal cord. Archives of Neurology 1986;43:121–5. Tsunoda I, Fujinami RS. Neuropathogenesis of Theiler’s murine encephalomyelitis virus infection, an animal model for multiple sclerosis. Journal of Neuroimmune Pharmacology 2010;5:355–69. Tuohy VK, Lu Z, Sobel RA, Laursen RA, Lees MB. Identification of an encephalitogenic determinant of myelin proteolipid protein for SJL mice. Journal of Immunology 1989;42:1523–7. Vanderluit JL, Bourque JA, Peterson AC, Tetzlaff W. Model for focal demyelination of the spinal dorsal columns of transgenic MBP-LacZ mice by phototargeted ablation of oligodendrocytes. Journal of Neuroscience Research 2000;62:28–39. Vandevelde M, Zurbriggen A. Demyelination in canine distemper virus infection: a review. Acta Neuropathologica 2005;109:56–68. Venturini G. Enzymatic activities and sodium, potassium and copper concentrations in mouse brain and liver after cuprizone treatment in vivo. Journal of Neurochemistry 1973;21:1147–51. Verbeek R, van Dongen H, Wawrousek EF, Amor S, van Noort JM. Induction of EAE by T cells specific for alpha B-crystallin depends on prior viral infection in the CNS. International Immunology 2007;19:277–85. Vereyken EJ, Fluitsma DM, Bolijn MJ, Dijkstra CD, Teunissen CE. An in vitro model for de- and remyelination using lysophosphatidyl choline in rodent whole brain spheroid cultures. Glia 2009;57:1326–40. Verwer RW, Hermens WT, Dijkhuizen P, ter Brake O, Baker RE, Salehi A, et al. Cells in human postmortem brain tissue slices remain alive for several weeks in culture. Faseb Journal 2002;16:54–60. Vesterinen HM, Sena ES, ffrench-Constant C, Williams A, Chandran S, Macleod MR. Improving the translational hit of experimental treatments in multiple sclerosis. Multiple Sclerosis 2010;16:1044–55. Wan L, Xia R, Ding W. Short-term low-frequency electrical stimulation enhanced remyelination of injured peripheral nerves by inducing the promyelination effect of brain-derived neurotrophic factor on Schwann cell polarization. Journal of Neuroscience Research 2010;88:2578–87. Wang Z, Colognato H, Ffrench-Constant C. Contrasting effects of mitogenic growth factors on myelination in neuron–oligodendrocyte co-cultures. Glia 2007;55: 537–45. Wilhelm I, Fazakas C, Krizbai IA. In vitro models of the blood–brain barrier. Acta Neurobiologiae Experimentalis (Wars) 2011;71:113–28. Williams A, Piaton G, Lubetzki C. Astrocytes—friends or foes in multiple sclerosis? Glia 2007;55:1300–12. Xiao J, Wong AW, Willingham MM, Kaasinen SK, Hendry IA, Howitt J, et al. BDNF exerts contrasting effects on peripheral myelination of NGF-dependent and BDNF-dependent DRG neurons. Journal of Neuroscience 2009;29:4016–22. Zamvil SS, Nelson PA, Mitchell DJ, Knobler RL, Fritz RB, Steinman L. Encephalitogenic T cell clones specific for myelin basic protein. An unusual bias in antigen recognition. Journal of Experimental Medicine 1985;162:2107–24. Zeis T, Kinter J, Herrero-Herranz E, Weissert R, Schaeren-Wiemers N. Gene expression analysis of normal appearing brain tissue in an animal model for multiple sclerosis revealed grey matter alterations, but only minor white matter changes. Journal of Neuroimmunology 2008;205:10–9. Zhang H, Jarjour AA, Boyd A, Williams A. Central nervous system remyelination in culture—a tool for multiple sclerosis research. Experimental Neurology, in press. Zhang Z, Drzewiecki GJ, Hom JT, May PC, Hyslop PA. Human cortical neuronal (HCN) cell lines: a model for amyloid beta neurotoxicity. Neuroscience Letters 1994;177:162–4. Zuiderwijk-Sick EA, van der Putten C, Bsibsi M, Deuzing IP, de Boer W, PersoonDeen C, et al. Differentiation of primary adult microglia alters their response to TLR8-mediated activation but not their capacity as APC. Glia 2007;55: 1589–600.

Uncovering cryptic glycan markers in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE).

Monoclonal Antibodies in Preclinical EAE Models of Multiple Sclerosis: A Systematic Review.

Multiple sclerosis-induced neuropathic pain: pharmacological management and pathophysiological insights from rodent EAE models.

Transcriptomic meta-analysis of multiple sclerosis and its experimental models.

Alemtuzumab in Multiple Sclerosis: Mechanism of Action and Beyond.

Acutely damaged axons are remyelinated in multiple sclerosis and experimental models of demyelination.

Immune surveillance of the central nervous system in multiple sclerosis--relevance for therapy and experimental models.

Genotoxicity of microcystin-LR in in vitro and in vivo experimental models.

Checklist for reporting and reviewing studies of experimental animal models of multiple sclerosis and related disorders.

Axonal pathology and demyelination in viral models of multiple sclerosis.

Inhibition of Drp1 hyper-activation is protective in animal models of experimental multiple sclerosis.

Synaptic plasticity in multiple sclerosis and in experimental autoimmune encephalomyelitis.

Oxidative tissue injury in multiple sclerosis is only partly reflected in experimental disease models.

Multiple Sclerosis and Experimental Allergic Encephalomyelitis.

Transcriptional dysregulation of Interferome in experimental and human Multiple Sclerosis.

Remitting-relapsing EAE in nonhuman primates: a valid model of multiple sclerosis.

in vivo chemically-induced experimental models of liver oxidative stress in rats.

Th Cell Diversity in Experimental Autoimmune Encephalomyelitis and Multiple Sclerosis.

Angiogenesis in multiple sclerosis and experimental autoimmune encephalomyelitis.

In vivo models of multiple myeloma (MM).

Theme 10 in vivo experimental models.

Theme 13 In vivo Experimental Models.

T reg balance in the EAE model of multiple sclerosis by targeting IL6.

Dalfampridine Effects Beyond Walking Speed in Multiple Sclerosis.