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between animals immunised with different batches of rmMOG. The absence of cortical pathology in this strain following induction of MOG-EAE supports previous findings (Storch et al., 2006). Interestingly, animals immunised with pXVII rmMOG did not develop cortical demyelination following subarachnoid injection of TNF and IFNγ, in contrast to subpial GMLs accompanied by microglial activation found in 3 of 4 animals pXIIIa-immunised rats. Since demyelination has been shown to be dependent on auto-antibodies in rat MOG-EAE (Linington and Lassmann, 1987; Piddlesden et al., 1993; Adelmann et al., 1995; Genain et al., 1995; Storch et al., 1998b; Iglesias et al., 2001), a further ELISA was performed to measure different antibody isotypes in peripheral blood from rats immunised with both rmMOG batches. Chapter 4 – Characterisation of acute subarachnoid targeted EAE 137

All groups had comparable levels of IgG2a, suggested to be a more pathogenic isotype than IgG1 due to the increased efficiency of complement fixation observed with IgG2a (Piddlesden et al., 1993). However IgG1 levels were observably reduced in rats immunised with 5μg pXVII compared to pXIIIa-immunised rats. Variation in demyelinating ability has been observed previously within the IgG1 isotype, which is suggested to be due to differences in affinities for MOG or the ability to bind macrophage Fc receptors (Piddlesden et al., 1993). The formation of WMLs in the spinal cord indicates that demyelinating antibodies are produced in response to immunisation with 50µg pXVII, but it is possible that immunisation with 5μg pXVII may have resulted in production of insufficient levels of pathogenic antibodies with a sufficient affinity to initiate demyelination following CNS entry during targeted EAE (Piddlesden et al., 1993). Production of antibodies with an insufficient affinity for MOG would decrease the persistence of antibody deposition on the myelin sheath and the duration of complement fixation and macrophage activation, and might not reach the threshold required for initiation of demyelination. Alternatively, it is possible that antibodies produced in response to immunisation with pXVII recognised a greater proportion of linear MOG epitopes, which have been shown not to directly participate in demyelination, compared to pathogenic conformational-dependent epitopes (Haase et al., 2001). Although the same isolation and purification protocol was followed in the production of both rmMOG batches, differences in the proportion of linear to conformational rmMOG species may have arisen between batches. Immunisation with bacterially expressed recombinant MOG is recognised to result in a complex anti-MOG antibody response, characterised by recognition of both linear and conformational epitopes (Adelmann et al., 1995; Litzenburger et al., 1998; Brehm et al., 1999; Iglesias et al., 2001). The use of urea in isolation protocols is suggested to denature and linearise the protein, resulting in differential processing of recombinant versus native proteins following immunisation in vivo (Iglesias et al., 2001). Measurement of IgG levels by ELISA does not necessarily indicate the presence of a demyelinating antibody response since ELISAs recognise antibodies specific for incorrectly folded and denatured MOG. Differences in linear versus conformational epitope specificities between animals immunised with different rmMOG batches in the current study would therefore not be detected and could explain the difference in pathology observed following subarachnoid injection of cytokines.

Fluorescent activated cell sorter (FACS) assays using cells transfected to express MOG can be used to confirm whether anti-MOG antibodies will recognise MOG expressed by cells in the CNS in vivo (Brehm et al., 1999; Haase et al., 2001), thus giving a better indication of Chapter 4 – Characterisation of acute subarachnoid targeted EAE 138

pathogenicity than ELISAs, but these assays are limited by the high variability of cell lines in use, highlighting the need for an ELISA which provides only correctly folded MOG as an antigen. A tetramer radioimmunoassay, where 4 MOG extracellular domains were tetramerised and used to detect antibodies via immunoprecipitation, was also found to be more specific than ELISA for detecting pathogenic anti-MOG antibodies. This was due to the increased avidity of the antigen and the more specific identification of antibodies against conformational, correctly folded MOG protein, as the MOG tetramer does not bind antibodies specific for linear MOG epitopes (O'connor et al., 2007).

Following the observation of subpial pathology in all 10μg pXIIIa rmMOG-immunised animals following cytokine injection, and no apparent difference in EAE incidence between the 5 or 10μg pXIIIa groups, it was concluded that 10μg pXIIIa was the optimum immunisation dose for use in further targeted EAE studies. No cortical demyelination or microglial activation was observed in animals at the peak of neurological deficit in either the 5 or 10μg pXIIIa groups, suggesting that in spite of spinal EAE pathology, cortical pathology following cytokine injection can still be reliably attributed to the effects of the injected cytokines. Changes in cortical NAGM gene expression have been observed in chronic EAE following immunisation with 50μg recombinant rat MOG in the absence of observable cortical pathology, in line with NAGM observations from MS studies (Dutta et al., 2006; Zeis et al., 2008). However these gene expression changes were not observed in animals with a variable EAE course similar to that of rats immunised with 5 or 10μg pXIIIa described in the current study, suggesting that chronic disease may be required to induce gene expression changes in cortical NAGM tissue distant from spinal lesion sites. Therefore, the 10μg pXIIIa rmMOG dose was used in future studies of the targeted EAE model to determine the effect of subarachnoid injection of LTα.

4.3.4 Alternative injection sites are less relevant to F+SPMS pathology