Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS). MS is also named encephalomyelitis disseminata (ED). EAE has been also called experimental allergic encephalomyelitis, but the word `allergic` is more and more replaced by the word `autoimmune`. This has to do with the deeper understanding of the disease biology that has been gathered over the last years. MS is a chronic autoimmune disease of the central nervous system (CNS) that leads to inflammation, demyelination and axonal loss. The CNS lesions cause neurological deficits.


Introduction
Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS).MS is also named encephalomyelitis disseminata (ED).EAE has been also called experimental allergic encephalomyelitis, but the word `allergic` is more and more replaced by the word `autoimmune`.This has to do with the deeper understanding of the disease biology that has been gathered over the last years.MS is a chronic autoimmune disease of the central nervous system (CNS) that leads to inflammation, demyelination and axonal loss.The CNS lesions cause neurological deficits.
In the beginning the disease course of MS is in most cases relapsing-remitting but changes after several years into a secondary chronic progressive disease course (Table 1).More rarely, there are also primary progressive disease courses.The diagnosis of MS is often preceded by a single demyelinating event for example the appearance of Optic Neuritis (ON).There are indications that MS is caused by the activation of autoreactive CNS-specific T cells and possibly antibodies that lead to subsequent activation of additional immune cascades and lesion formation (Fig. 1).The relapsing phase of MS is thought to be predominately mediated by the adaptive phase of the immune response, while progressive forms are more driven by innate immune mechanisms (Bhat and Steinman, 2009;Weiner, 2009).
The EAE models that are used in the laboratory for assessment of immunological, neurobiological and therapeutic studies are all induced models or genetically modified models.There is no naturally occurring spontaneous EAE model that is accepted as a valuable laboratory model for MS.EAE can be induced in various animal strains and species.Most used species are mice and rats.The reason for this is the size, the availability of inbred strains and the possibility for genetic modification as well as the immense number of tools to characterize rodents.In addition certain monkey species like marmosets are used for specific questions that cannot be easily assessed in rodents (Hart et al., 2011).
What has become clear over the last years is the fact that EAE is no perfect model for all aspects of MS.Rather various different models represent facets of MS.Some models are more suited for immunological analyses and this can be further divided into adaptive and innate immunity related aspects as well as cellular aspects, like the analysis of influences of T and B cells on disease precipitation and maintenance.There are models that are more suited for analysis of certain neurobiological aspects of the disease, like axonal and neuronal pathology and detailed lesion characterization.MS variants can be modeled in rodents.For example Acute Disseminated Encephalomyelitis (ADEM), an acute inflammatory reactive disease of the CNS, and Neuromyelitis Optica (Devic`s disease) can be modeled in certain rodent species as outlined further below.The researcher who wants to use EAE as a model should be aware of all the various types of EAE and should be careful in selecting the correct species, strain and immunization protocol, since depending on these factors, the outcome and interpretability of the research will differ.The selection of the specific model might differ strongly between an immunologist who wants to assess basic principles of organ specific autoimmunity in contrast to the MS researcher who wants to analyze specific disease aspects or a new therapeutic approach in preclinical pharmacology studies.(Fissolo et al., 2009).On the ground of a genetic predisposition with the possible cofactors vitamin D and sunlight exposure the disease is induced and autoimmunity is maintained by aberrant immune cascades that will finally lead to myelin/oligodendrocyte pathology as well as axonal and neuronal loss.There are rodent EAE models that can be induced by passive transfer of T cells.These models are not subject of this chapter and are therefore not outlined in detail.T cell transfer models have been very suitable for immunological investigations mainly in regard to the dissection of the role of T cells in organ-specific autoimmunity (Krishnamoorthy and Wekerle, 2009).

Establishment of Myelin-Oligodendrocyte-Glycoprotein (MOG)-induced EAE in rats
The rat is a good species to perform EAE studies due to its larger size as compared to mice, the availability of many well characterized inbred strains, its strong standing for pharmacological studies and its great value for behavioral outreads.In addition novel ways for genetic modification are increasingly becoming available.The rat Major Histocompatibility Complex (MHC) is called RT1.The classical MHC I molecule is called RT1.A, the MHC II molecules are named RT1.B and RT1.D that are equivalent to HLA-DQ (RT1.B) and HLA-DR (RT1.D) (Fig. 2, Table 3).
EAE induced with whole myelin extracts or MBP in LEW (RT1 l ) rats has been used over many years (Table 2).This model is monophasic and was the prototype EAE model in the past.Much understanding regarding MS pathogenesis, immunology and neurobiology has been obtained over the years.The model is very useful to study basic immunological principles regarding T cell migration to the CNS and T cell related immunity of MS.This Experimental Autoimmune Encephalomyelitis -Models, Disease Biology and Experimental Therapy 8 model is often used for pharmacological investigations in EAE.Beside with whole myelin extracts the model can be induced with MBP protein (various isoforms) or peptides from the main encephalitogenic regions MBP 89-101 and MBP 68-88.Interestingly MBP peptide 89-101 binds to RT1.D l (HLA-DR-like), while MBP 68-88 binds to RT1.B l (HLA-DQ-like) (de Graaf et al., 1999;Weissert et al., 1998a).There are species dependent effects of MBP on disease development.We could demonstrate that the MBP peptide 63-88 derived from Guinea Pig (GP) as compared to the MBP peptide 63-88 derived from rat is much more encephalitogenic.Immunization with MBP GP 63-88 results in the agglomeration of T cells in the CNS that express up to 30% the TCRBV chain 8.2 (TCRBV8S2), while after immunization with MBP RAT 63-88 there is not such a strong overusage of such T cells in the CNS (Weissert et al., 1998a).Based on this fact it can also be understood that therapeutic manipulation in the context of immunization with MBP GP 63-88 is more demanding as compared to MBP RAT 63-88.We equally demonstrated that MBP derived peptides of different species can act as superagonists, agonists or antagonists depending on the expressed MHC II haplotype ( de Graaf et al., 2005).3. RT1 haplotypes of inbred rat strains (Weissert et al., 1998b).RT1.B is the rat equivalent to HLA-DQ and RT1.D to HLA-DR.*Donor strain in brackets It was demonstrated that as compared to LEW rats, DA rats develop in a much higher incidence chronic disease after immunization with autologous whole spinal cord www.intechopen.comhomogenates (Lorentzen et al., 1995).In DA rats immunization of autologous whole spinal cord in IFA is sufficient to induce this type of chronic disease.Interestingly it was found that DA rats develop antibodies against the MOG extracellular domain MOG 1-125.Based on this fact we were interested to assess the encephalitogenic potential of MOG 1-125 in DA rats and other inbred rat strains.We found that immunization with MOG 1-125 in CFA resulted in relapsing-remitting disease (Weissert et al., 1998b).Also the immunization of MOG 1-125 in IFA causes to this type of disease.Interestingly for the first time it was observed in rodents that the rats developed ON in addition to classical EAE symptoms (Storch et al., 1998).In addition, specific immunization protocols allowed the selective induction of ON.ON is a typical aspect of MS.The CNS lesions in this model have much greater similarity to MS as compared to the lesions in LEW rats immunized with MBP or MBP peptides.Widespread demyelination was present and the analysis of lesions resulted in the important insight that beside T cells also antibodies contribute to lesion formation in this model.In contrast to MBP, MOG is expressed on the exterior part of the myelin sheath and the Ig-like domain can be recognized by antibodies.Binding of antibodies to MOG can activate a number of immune mechanisms like the activation of macrophages and complement deposition that can result in increased tissue damage as compared to purely T cell mediated pathology.

Strain
Based on the findings in DA rats and immunization with MOG we were wondering about the encephalitogenic potential in rat strains with different MHC haplotypes.This question appeared important to us, since it has been demonstrated for a long time that the MHC II region has a strong genetic influence on MS (Sawcer et al., 2011).Therefore we used a large number of inbred congenic LEW rat strains that express a wide variety of MHC haplotypes.
We observed that certain rat strains developed relapsing-remitting or chronic progressive disease, others hyperacute progressive disease, some slow progressive disease with predominance of cortical pathology and others were protected (Table 4).Also the selection of the adjuvant had additional effects.While LEW.1N (RT1 n ) immunized with MOG 1-125 in CFA developed hyperacute progressive disease that often lead to death due to pontine lesions, immunization with MOG 1-125 in IFA resulted in chronic progressive disease with axonal pathology that has great similarity to MS (Kornek et al., 2000).
We also observed that LEW (RT1 l ) rats immunized with MOG 1-125 did not develop EAE (Weissert et al., 1998b).This is in contrast to immunization protocols with MBP or MBP peptides which lead to EAE as outlined above (Weissert et al., 1998a).Based on the fact that LEW.1AV1 rats that carry the RT1 av1 haplotype derived from the DA rat develop EAE after immunization with MOG 1-125, we concluded that the MHC haplotype is operating in the context of the myelin antigen used for immunization (Weissert et al., 1998b).This was further supported by the observation that LEW.1N (RT1 n ) rats and BN (RT1 n ) rats do not develop disease after immunization with MBP, but strong disease after immunization with MOG 1-125.This finding appears of some importance since it might explain why MHC haplotypes might differ as susceptibility loci in different parts of the world: depending on the environmental challenges that differ in different regions of the world, the subsequent induction of immunity against certain myelin components might differ.For example in Western Europe and North America the main susceptibility HLA allele is HLA-DR2b, while in Sardinia it is HLA-DR4 (Marrosu et al., 1997).
Experimental Autoimmune Encephalomyelitis -Models, Disease Biology and Experimental Therapy 10 We described rat strains that carried MHC haplotypes that allow disease development after immunization with MOG 1-125 but which were protected by non-MHC genomes.We defined that ACI rats as well as MHC congenic PVG-RT1 av1 that both carry the RT1 av1 haplotpye were protected from EAE (Weissert et al., 1998b).Subsequent large genetic screens allowed dissection of some of the susceptibility genes (Swanberg et al., 2005).
We found one LEW rat strain with a specific MHC haplotype that predominantly develops cortical lesions, LEW.1AR1 (RT1 r2 ) rats (Storch et al., 2006;Weissert et al., 1998b).Recently cortical lesions have been acknowledged as a primary cause of disability in MS.Cortical lesions can be also induced in LEW rats with a subencephalitogenic immunization with myelin components and subsequent local intrathecal application of Tumor Necrosis Factor alpha (TNF) (Merkler et al., 2006).Marmosets immunized with MOG 1-125 can develop cortical lesions as well (Pomeroy et al., 2005).By now it has also been observed that certain mouse strains can develop cortical pathology (Mangiardi et al., 2011).In our observation LEW.1AR1 rats immunized with MOG 1-125 in CFA represent the most suitable and reproducible model for cortical pathology of MS and possible therapeutic manipulation.
I n a n e x t s t e p w e w e r e i n te r e s t e d t o d e f i ne the encephalitogenic stretches within the extracellular domain of MOG 1-125 (Tables 5, 6).In order to do this we used 18 amino acid long overlapping peptides of the MOG 1-125 rat sequence (Weissert et al., 2001).We performed this study in many different inbred and inbred MHC congenic rat strains.Interestingly we found that only MOG 91-108 in CFA induced disease in rat strains with the RT1 av1 or RT1 n haplotype.Rat strains with the RT1 av1 haplotype are DA rats and LEW.1AV1 rats and rats with the RT1 n haplotype LEW.1N and BN rats.We found that MOG 91-108 bound well to purified RT1.B a molecules and to RT1.D n molecules.
Immunization with MOG 91-108 induced a T and B cell response.In LEW.1N rats this T cell response was difficult to measure and we concluded early that other factors than classical Th1 mediated cytokines might be operative contributing to disease precipitation (Weissert et al., 2001).
In most instances the immunodominant peptides did not correspond to the peptides that were capable of inducing EAE.We concluded that the immune response to the immunodominant peptides might be also a signature of a regulatory T cell response (we called it `modulatory).Table 5. MOG peptides that induce disease and immune responses.Most MOG stretches that induce strong immune responses in rats do not induce EAE (Weissert et al., 2001).Monophasic or chronic (de Graaf et al., 2008) Table 6.EAE models induced with MOG peptides.Based on detailed immunological analysis the region MOG 91-108 was defined as the encepathalitogenic region in different rat strains.Further dissection allowed the narrowing of the disease inducing MOG stretches to nine amino acid long peptides.

RT1
We observed that complement depletion does lead to protection from disease, underscoring the influence of antibodies to MOG.Crystallographic studies have indicated that the region MOG 91-108 is accessible to antibodies (Breithaupt et al., 2008).In addition, we demonstrated by spectroscopic TCR analysis that depending on the expressed RT1 haplotype, the predominance of certain TCRBV chains was the same in rat strains with different non-MHC genomes underscoring the strong influence of the MHC II haplotype on TCRBV usage in the MOG model (de Graaf et al., 2008).
For the rat EAE models, we measured the binding strength of the encephalitogenic peptides by comptetitive binding assays (Table 7).We could show that the peptides that induce EAE are binding well to the MHC II molecules that present the peptides to T cells ( de Graaf et al., 2008;de Graaf et al., 1999;Weissert et al., 2001;Weissert et al., 1998a).We dissected the binding qualities to purified RT1.B and RT1.D molecules and the T and B cell response to MOG 91-108 in LEW.1N and LEW.1AV1 rats.We found that the peptides that bound strongest, induced EAE.This were the peptides MOG 96-104 in LEW.1AV1 rats binding to RT1.B a and MOG 98-106 in LEW.1N rats binding to RT1.D n .With increasing shortening of the peptides, the evolving disease was partly reduced, indicating that possibly there was a reduction in the activated encephalitogenic T cell repertoire ( de Graaf et al., 2008).
In summary we made a major step in establishing more suitable models to study MS disease biology and therapeutic interventions.In addition we were able to obtain a large quantity of insight into the immune regulation in the context of genetic factors in a complex autoimmune disease.We demonstrated the major influence of MHC II haplotypes on disease regulation, but for the first time also of MHC I.Recently also for MS influences of MHC I loci on susceptibility could be confirmed (Sawcer et al., 2011).

Ways of EAE induction with either MBP, PLP or MOG and choice of the relevant animal model/strain
Beside the selection of the right species and strain, the selection of the model antigen is of great importance for the outcome of the EAE studies.The sequences of the most used Experimental Autoimmune Encephalomyelitis -Models, Disease Biology and Experimental Therapy 14 stretches of myelin proteins for disease induction used in different species are listed in Table 8.The peptides should have a high degree of purity and it is advisable to prepare large batches that can be used over long term for experimentation.In the case of EAE induction with recombinant antigens, also larger scale preparation and usage of identical batches over longer time is recommendable, since there is the danger of batch to batch variation that can dramatically affect the outcome of experimentation.
In addition to species, strain and antigen, the adjuvant is of major importance for the success of the EAE induction and the outcome of the experimentation (Table 9).While in the rat models, the usage of IFA or CFA is sufficient, nearly all mouse models require the addition of PT in the immunization protocol and booster immunizations.The preparation of the antigen/adjuvant mixture requires much care and the presence of a homogenate emulsion is needed for successful immunizations.
In the past immunizations have often been performed in the foot pads of the rodents.Due to obvious ethical reasons, this procedure is not any more applied.In addition this procedure results in the swelling of the footpads affecting gait.This type of gait disturbance can blur EAE symptoms with the consequence of wrongly reported EAE scores.Nowadays, in rats the immunization is done as a single injection in the base of the tail, while in mice multiple injections in the flanks are used for the procedure.It is advisable to establish the best suited immunization protocol in the laboratory with care, after the selection of the model based on scientific rationales has been performed.(de Graaf et al., 2008;Weissert et al., 2001) Table 9. Immunization regimen for different EAE models and usage of adjuvants.CFA = Complete Freund`s Adjuvant; IFA = Incomplete Freund`s Adjuvant; PT = Pertussis Toxin.

Conclusions
It is well possible to induce different aspects of MS in rodent EAE models (Table 10).Some models are more suited for immunological analysis, while others better serve the neuroscience community.None of the models can model all aspects of MS.Based on the specific scientific question, the most suitable EAE model for a specific analysis should be selected based on the characteristics of the model.The selection of the best suited model will result in better results of the overall research project and will improve the interpretability of the results.

Course of MS Type of EAE Strain Immunization
Relapsing The MOG-EAE model in rats with the availability of various inbred and RT1 congenic strains provides a very good and well defined system for assessment of pertinent questions regarding MS disease biology and therapeutic interventions.

Fig. 1 .
Fig. 1.Possible cascade for induction of MS.So far it is not clear what induces MS.Trigger factors are possibly infections, adjuvants or the endogenous presentation of self antigens(Fissolo et al., 2009).On the ground of a genetic predisposition with the possible cofactors vitamin D and sunlight exposure the disease is induced and autoimmunity is maintained by aberrant immune cascades that will finally lead to myelin/oligodendrocyte pathology as well as axonal and neuronal loss.

Table 1
. Different disease courses of MS.Relapsing disease courses of MS are mainly driven by the adaptive immune response (T and B cells) while progressive disease is thought to be predominantly mediated by innate immunity.In most cases, relapsing disease changes over the course of MS to secondary progressive disease.As outlined in Table10, specific EAE models can be used to mimic the aspects of different types and variants (Devic´s, ADEM) of MS.

Table 2 .
Presently most used EAE models for laboratory research.The list only provides a part of all available EAE models.CFA = Complete Freund`s Adjuvant; IFA = Incomplete Freund`s Adjuvant; PT = Pertussis Toxin.modified models include the spontaneous EAE in C57BL/6 mice that have a T Cell Receptor (TCR) specific for MBP peptide 1-9.Another genetically modified C57BL/6 mouse with a MOG specific TCR and a MOG specific B Cell Receptor (BCR) or surface Immunoglobulin (sIg) develop spontaneous EAE and can be very useful for assessment of specific scientific questions.

Table 8 .
Sequences of myelin peptides used for EAE induction in mice and rats

Table 10 .
Best suited rat model to investigate aspects of different MS types and MS variants