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Multiple sclerosis is an inflammatory and neurodegenerative autoimmune disease of the central nervous system that presents with perivenous lymphocytic infiltration, focal demyelination and neuro-axonal degeneration. Neuro-axonal injury is a key contributor to non-reversible long-term disability in multiple sclerosis, however, the exact mechanisms underlying neurodegeneration as well as the sequence of events leading to structural and functional impairment are not yet fully understood. Visual impairment, including optic neuritis, is a common and early clinical feature of multiple sclerosis, with up to 75% of patients experiencing some form of visual disability during their disease course. Frequently, episodes of multiple sclerosis related acute optic neuritis are followed by structural retinal damage that can be quantified in vivo using optical coherence tomography (OCT). Reduced retinal peripapillary nerve fibre layer and macular ganglion cell layer thickness assessed by OCT have been shown to correlate strongly with functional visual impairment and- on a broader level - brain atrophy among multiple sclerosis patients. Similar to findings in human studies, alterations in the retina including the retinal ganglion cells and optic nerve have been reported in a mouse model of multiple sclerosis called experimental autoimmune encephalomyelitis (EAE). However, the exact biological basis and temporal dynamics behind the retinal abnormalities observed clinically and in OCT studies, including the complex interplay between inflammatory and degenerative mechanisms have not yet been fully elucidated. EAE can be used to investigate structural damage in the afferent visual pathway, providing a unique model for assessing neurodegenerative changes and their functional consequences. In addition, it has the potential to generate deeper insight into basic mechanisms underlying structural tissue damage in multiple sclerosis. To assess visual pathway damage in EAE, an OCT and magnetic resonance imaging platform was developed and established. OCT detected inner retinal layer thickness changes in EAE mice, resembles what is observed in human multiple sclerosis related acute papillitis. Where an initial phase of swelling was associated with inner retinal layer thickening, followed by a significant decrease in thickness over time, once the swelling had subsided, likely representative of neuro-axonal pathology. Diffusion tensor imaging measurements of the optic nerve and tract provided complementary results suggestive of demyelination and axonal damage in the visual pathway. OCT retinal thickness also correlated significantly with diffusion tensor imaging measurements providing support for retrograde and anterograde degeneration in the visual pathway following optic neuritis. Immunofluorescence analysis contributed further evidence for a strong inflammatory response in the retina and optic nerve in EAE mice at the final observational time point. A combination of both OCT and magnetic resonance imaging provided a reliable and sensitive quantitative tool to assess structural damage in the visual pathway and the temporal sequence of neurodegeneration in EAE. To further characterize the biological mechanisms of visual pathway impairment in EAE and to unravel the morphological correlates of retinal thickness changes, a longitudinal OCT study including a systematic assessment of retinal and optic nerve immunohistochemical analysis was performed. Signs of inflammatory edema 11 days post immunisation coincided with inner retinal layer thickening, while neuro-axonal degeneration throughout the disease course contributed to inner retinal layer thinning observed at later time points. Early retinal pathology, including axonal transport impairment, was observed prior to cellular infiltration (i.e. T-cells) in the optic nerve 11 days post immunisation. However, the consequences of early retinal damage on OCT-derived measurements were offset by the initial inflammatory edema. Microgliosis and astrocytosis was detected in the retina prior to optic neuritis and persisted until the final observational time point. Early glial activity likely contributed to initial signs of retinal pathology that appeared in the absence of cellular infiltration, suggesting a need for early intervention of optic neuritis. Subsequent to inflammation, Müller cells responded to retinal pathology with possible neuroprotective behaviour. Müller cell reactivity (i.e. aquaporin-4 and glutamine synthetase decrease) occurred after 11 days post immunisation in the inner retinal layer. Future studies should explore Müller cell reactivity and its potentially neuroprotective role. Severe neuro-axonal degeneration was observed in the optic nerve and retina until 33 days post immunisation. Although, retrograde degeneration likely promoted the majority of observed inner retinal layer damage following optic neuritis, primary pathology – possibly due to gliosis – also contributed to inner retinal layer thinning. These results added morphological substrate to the OCT findings, further solidifying this tool as a valuable method to assess visual pathway damage in EAE and multiple sclerosis. Although, retinal ganglion cell pathology had been observed using immunohistochemical analysis and corroborated inner retinal layer thickness findings, it is still unclear to what extent retinal ganglion cell loss plays a role in OCT observed thinning of the retina. In vivo measurements of retinal ganglion cells can provide additional information on the time-course of neuro-axonal pathology within the same mouse, which histology is not capable of. A transgenic mouse line with yellow fluorescent proteins expressed in the Thy1 sequence (a marker for neurons including retinal ganglion cells) was used in conjunction with green autofluorescence imaging to examine neuro-axonal damage in the retina longitudinally. Feasibility and longitudinal reliability of green autofluorescence imaging to assess yellow fluorescent protein expressing cells was confirmed in healthy transgenic mice. To measure the number of yellow fluorescent protein expressing cells in the retina both a manual and automated cell counting method was employed. Although the automated cell counting method had high variance compared to the manual cell count, it was significantly faster and allowed for a greater area on the fundus image to be examined, giving a more unbiased representative read-out of neurodegeneration and allowing for spatial statistics. In EAE mice, hyperintensity of the yellow fluorescent signal was observed around the optic nerve head at onset of clinical symptoms, likely coinciding with the occurrence of optic neuritis in this model. Interestingly, this phenomenon dissipated at peak of symptoms in almost all the EAE mice. Green autofluorescence imaging may be a useful way to identify the occurrence of optic neuritis in EAE through the hyperintensity observed at clinical onset of symptoms, especially compared to standard magnetic resonance imaging or ex-vivo methods of confirming optic neuritis. Future studies should evaluate the value of this model for assessing retinal ganglion cell death or neuropathology at later stages of experimental optic neuritis. The EAE induced Thy1 transgenic mouse model would be valuable in future pre-clinical trials that want to target retinal ganglion cells. Overall, investigating structural anterior visual pathway damage may constitute a unique model for assessing mechanism and temporal sequence of neurodegeneration in multiple sclerosis. The extent and rapid onset of axonal and neuronal damage in this model appears relevant for pre-clinical trials and for testing interventions scaled to multiple sclerosis.

Neuromyelitis optica (NMO) and multiple sclerosis (MS) both result in acute injury (i.e. attacks or relapses) to the central nervous system with focal demyelination and axonal loss that varies in severity along a spectrum. A variety of non-invasive structural imaging and functional tools can be used to investigate mechanisms of white matter injury and secondary axonal injury in MS and NMO. These include advanced magnetic resonance imaging (MRI) measures of myelin water fraction; optical coherence tomography (OCT) for retinal nerve fibre layer thickness and total macular volume; and transcranial magnetic stimulation (TMS) to determine cortical excitability and integrity of cortical spinal pathways. First, the relationship between a functional measure using TMS and a structural measure of myelin in the cortico-spinal tract was examined. Structural changes were found in the descending motor output pathway white matter in NMO along with abnormal TMS measures, suggesting that there is greater spinal cord involvement and more extensive axonal loss found in NMO compared to MS. Next, OCT was used as a measure of the anterior visual pathway and myelin water imaging of the posterior visual pathway; the effects of damage to one part of the visual system on the other was studied. Retrograde degeneration to the retina and anterograde degeneration to the optic radiations from the optic nerve was observed in both MS and NMO subjects with optic neuritis history. A correlation between the measures indicating that damage to one part may cause damage to another part of the visual pathway. Finally, damage was observed in optic pathway in MS patients without optic neuritis history suggesting that there is damage in the absence of lesions in the optic nerve. Finally, myelin water imaging was used to investigate if the disease burden of lesions regulate the level of damage to the normal appearing white matter (NAWM) tracts. The lack of correlation between disease burden of lesions and NAWM myelin water imaging in MS suggested that damage to the NAWM was mediated by processes independent of lesions. These techniques can be used to study and better understand demyelinating diseases such as MS and NMO.

The exact mechanisms and temporal sequence of neurodegeneration in multiple sclerosis are still unresolved. The visual pathway including its unmyelinated retinal axons, can serve as a prototypic model of neurodegeneration in experimental optic neuritis. We conducted a longitudinal study combining retinal imaging through optical coherence tomography (OCT) with immunohistochemical analyses of retinal and optic nerve tissue at various time points in experimental autoimmune encephalomyelitis (EAE). Inner retinal layer (IRL) thickness was measured in 30 EAE and 14 healthy control C57BL/6 J mice using OCT. Distribution of marker proteins was assessed by immunofluorescence staining and retinal mRNA levels were assayed using real-time PCR. Histological morphology was evaluated on light and electron microscopy images. Signs of inflammatory edema 11 days post immunisation coincided with IRL thickening, while neuro-axonal degeneration throughout the disease course contributed to IRL thinning observed after 20 days post immunisation. Retinal pathology, including axonal transport impairment, was observed early, prior to cellular infiltration (i.e. T-cells) in the optic nerve 11 days post immunisation. Yet, the effects of early retinal damage on OCT-derived readouts were outweighed by the initial inflammatory edema. Early microglial activation and astrocytosis was detected in the retina prior to retinal ganglion cell loss and persisted until 33 days post immunisation. Müller cell reactivity (i.e. aquaporin-4 and glutamine synthetase decrease) presented after 11 days post immunisation in the IRL. Severe neuro-axonal degeneration was observed in the optic nerve and retina until 33 days post immunisation. Initial signs of retinal pathology subsequent to early glial activity, suggests a need for prophylactic treatment of optic neuritis. Following early inflammation, Müller cells possibly respond to retinal pathology with compensatory mechanisms. Although the majority of the IRL damage observed is likely due to retrograde degeneration following optic neuritis, initial pathology, possibly due to gliosis, may contribute further to IRL thinning. These results add morphological substrate to our OCT findings. The extent and rapid onset of axonal and neuronal damage in this model appears relevant for testing interventions scaled to human optic neuritis., Acta Neuropathologica Communications, 7 (1), ISSN:2051-5960

Acta Neuropathologica Communications, 7 (1), ISSN:2051-5960

The exact mechanisms and temporal sequence of neurodegeneration in multiple sclerosis are still unresolved. The visual pathway including its unmyelinated retinal axons, can serve as a prototypic model of neurodegeneration in experimental optic neuritis. We conducted a longitudinal study combining retinal imaging through optical coherence tomography (OCT) with immunohistochemical analyses of retinal and optic nerve tissue at various time points in experimental autoimmune encephalomyelitis (EAE). Inner retinal layer (IRL) thickness was measured in 30 EAE and 14 healthy control C57BL/6 J mice using OCT. Distribution of marker proteins was assessed by immunofluorescence staining and retinal mRNA levels were assayed using real-time PCR. Histological morphology was evaluated on light and electron microscopy images. Signs of inflammatory edema 11 days post immunisation coincided with IRL thickening, while neuro-axonal degeneration throughout the disease course contributed to IRL thinning observed after 20 days post immunisation. Retinal pathology, including axonal transport impairment, was observed early, prior to cellular infiltration (i.e. T-cells) in the optic nerve 11 days post immunisation. Yet, the effects of early retinal damage on OCT-derived readouts were outweighed by the initial inflammatory edema. Early microglial activation and astrocytosis was detected in the retina prior to retinal ganglion cell loss and persisted until 33 days post immunisation. Müller cell reactivity (i.e. aquaporin-4 and glutamine synthetase decrease) presented after 11 days post immunisation in the IRL. Severe neuro-axonal degeneration was observed in the optic nerve and retina until 33 days post immunisation. Initial signs of retinal pathology subsequent to early glial activity, suggests a need for prophylactic treatment of optic neuritis. Following early inflammation, Müller cells possibly respond to retinal pathology with compensatory mechanisms. Although the majority of the IRL damage observed is likely due to retrograde degeneration following optic neuritis, initial pathology, possibly due to gliosis, may contribute further to IRL thinning. These results add morphological substrate to our OCT findings. The extent and rapid onset of axonal and neuronal damage in this model appears relevant for testing interventions scaled to human optic neuritis. Electronic supplementary material The online version of this article (10.1186/s40478-019-0768-5) contains supplementary material, which is available to authorized users.