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Abstract

Optic neuritis (ON) is a common early symptom in multiple sclerosis (MS). It is characterized by disruption of the blood-brain barrier (BBB), inflammatory demyelination, and reactive gliosis in addition to axonal and somatic degeneration of retinal ganglion cells (RGCs) within the optic nerve and retina, respectively. ON can be modelled in animals, in particular by immunization of Brown Norway (BN) rats with myelin oligodendrocyte glycoprotein (MOG). In addition to the hallmarks of ON, RGC degeneration in BN autoimmune optic neuritis (AON) has been reported to begin during the induction phase of the disease (iAON) prior to lymphocyte infiltration and inflammatory demyelination in the optic nerve (Fairless at al., 2012). This may have parallels with observations of retinal degeneration in some MS patients even in the absence of clinically defined ON associated with demyelination (Green at al., 2010; Saidha et al., 2011). Previous studies from our group have shown that this early RGC loss is timed with blood-retinal barrier (BRB) disruption, increase in retinal Ca2+ and activation of the Ca2+-activated protease, calpain (Hoffmann et al., 2013), with subsequent elevation of optic nerve Ca2+. We hypothesise that an initial retinal pathology may lead to Ca2+-mediated cytoskeleton reorganization and axonal transport deficits in the optic nerve that could affect axonal integrity, as observed during iAON by electron microscopy (Fairless et al., 2012). In this study, we examined the potential accumulation of axonal transport proteins along optic nerves, with particular focus on the optic nerve head (ONH) where RGC axons are naturally unmyelinated and therefore potentially more exposed to stress. No such anomalies were observed prior to the appearance of inflammatory demyelinating lesions. However, the axonal actin network dynamics within the optic nerve was changed starting iAON. This correlated with a decrease in protein expression of the Ca2+-dependent actin binding protein gelsolin, and an increase in fractin, a cleaved product of the actin monomer; both proteins being implicated in apoptotic processes. Intravitreal injection (IVI) of MK801, an NMDA receptor blocker, during iAON significantly rescued RGCs and reverted axonal actin changes to the values observed in healthy animals. In order to investigate downstream mechanisms of NMDA activity, a primary RGC culture was established. Based on qPCR data that Na+/Ca2+ exchanger 1 (NCX1), involved in regulating Ca2+ homeostasis, is elevated during iAON, its role in Ca2+ dynamics of isolated RGCs was investigated. Although this primary culture did not fully reflect RGC in vivo pathology (for example, cultured RGCs were seen not to retain sensitivity to glutamate excitotoxicity), blockade with an inhibitor targeting reverse NCX1 function (SEA0400) significantly reduced Ca2+ responses to glutamate stimulation. However, SEA0400 treatment during iAON in vivo did not affect either RGC survival or optic nerve actin dynamics. Collectively, data presented in this thesis provide further evidence that early neurodegenerative processes involved in AON pathology precede inflammatory driven demyelination of the optic nerves. Initial RGC loss observed in iAON is tightly connected to NMDA receptor activity in the retina, whereby prolonged intracellular Ca2+ increase may be one of the possible initiators of RGC death. These findings could be relevant for the development of potential neuroprotective treatment strategies in MS patients, particularly for a sub-set of MS patients characterized by retinal pathology in the absence of clinically-defined ON.