Chronic disability in multiple sclerosis is linked to neuroaxonal degeneration. 4-aminopyridine (4-AP) is used and licensed as a symptomatic treatment to ameliorate ambulatory disability in multiple sclerosis. The presumed mode of action is via blockade of axonal voltage gated potassium channels, thereby enhancing conduction in demyelinated axons. In this study, we provide evidence that in addition to those symptomatic effects, 4-AP can prevent neuroaxonal loss in the CNS. Using in vivo optical coherence tomography imaging, visual function testing and histologic assessment, we observed a reduction in retinal neurodegeneration with 4-AP in models of experimental optic neuritis and optic nerve crush. These effects were not related to an anti-inflammatory mode of action or a direct impact on retinal ganglion cells. Rather, histology and in vitro experiments indicated 4-AP stabilization of myelin and oligodendrocyte precursor cells associated with increased nuclear translocation of the nuclear factor of activated T cells. In experimental optic neuritis, 4-AP potentiated the effects of immunomodulatory treatment with fingolimod. As extended release 4-AP is already licensed for symptomatic multiple sclerosis treatment, we performed a retrospective, multicentre optical coherence tomography study to longitudinally compare retinal neurodegeneration between 52 patients on continuous 4-AP therapy and 51 matched controls. In line with the experimental data, during concurrent 4-AP therapy, degeneration of the macular retinal nerve fibre layer was reduced over 2 years. These results indicate disease-modifying effects of 4-AP beyond symptomatic therapy and provide support for the design of a prospective clinical study using visual function and retinal structure as outcome parameters.

Background: Retinal optical coherence tomography (OCT) is a clinical and research tool in multiple sclerosis, where it has shown significant retinal nerve fiber (RNFL) and ganglion cell (RGC) layer thinning, while postmortem studies have reported RGC loss. Although retinal pathology in experimental autoimmune encephalomyelitis (EAE) has been described, comparative OCT studies among EAE models are scarce. Furthermore, the best practices for the implementation of OCT in the EAE lab, especially with afoveate animals like rodents, remain undefined. We aimed to describe the dynamics of retinal injury in different mouse EAE models and outline the optimal experimental conditions, scan protocols, and analysis methods, comparing these to histology to confirm the pathological underpinnings.Methods: Using spectral-domain OCT, we analyzed the test-retest and the inter-rater reliability of volume, peripapillary, and combined horizontal and vertical line scans. We then monitored the thickness of the retinal layers in different EAE models: in wild-type (WT) C57Bl/6J mice immunized with myelin oligodendrocyte glycoprotein peptide (MOG 35-55 ) or with bovine myelin basic protein (MBP), in TCR 2D2 mice immunized with MOG 35-55 , and in SJL/J mice immunized with myelin proteolipid lipoprotein ). Strain-matched control mice were shamimmunized. RGC density was counted on retinal flatmounts at the end of each experiment.Results: Volume scans centered on the optic disc showed the best reliability. Retinal changes during EAE were localized in the inner retinal layers (IRLs, the combination of the RNFL and the ganglion cell plus the inner plexiform layers). In WT, MOG 35-55 EAE, progressive thinning of IRL started rapidly after EAE onset, with 1/3 of total loss occurring during the initial 2 months. IRL thinning was associated with the degree of RGC loss and the severity of EAE. Sham-immunized SJL/J mice showed progressive IRL atrophy, which was accentuated in PLP-immunized mice. MOG 35-55 -immunized TCR 2D2 mice showed severe EAE and retinal thinning. MBP immunization led to very mild disease without significant retinopathy.(Continued on next page)

Many biomarkers in clinical neuroscience lack pathological certification. This issue is potentially a significant contributor to the limited success of neuroprotective and neurorestorative therapies for human neurological disease – and is evident even in areas with therapeutic promise such as myelin repair. Despite the identification of promising remyelinating candidates, biologically validated methods to demonstrate therapeutic efficacy or provide robust preclinical evidence of remyelination in the central nervous system are lacking. Therapies with potential to remyelinate the central nervous system constitute one of the most promising and highly anticipated therapeutic developments in the pipeline to treat multiple sclerosis and other demyelinating diseases. The optic nerve has been proposed as an informative pathway to monitor remyelination in animals and human subjects. Recent clinical trials using visual evoked potential (VEP) have had promising results, but without unequivocal evidence about the cellular and molecular basis for signal changes on VEP, the interpretation of these trials is constrained. The VEP was originally developed and utilized in the clinic as a diagnostic tool but its use as a quantitative method for assessing therapeutic response requires certification of its biological specificity. Here, using the tools of experimental pathology we demonstrate that quantitative measurements of myelination using both histopathological measures of nodal structure and ultrastructural assessments correspond to VEP latency in both inflammatory and chemical models of demyelination. VEP latency improves after treatment with a tool remyelinating compound (clemastine), mirroring both quantitative and qualitative myelin assessment. Furthermore, clemastine does not improve VEP latency following demyelinating injury when administered to a transgenic animal incapable of forming new myelin. Therefore, using the capacity for therapeutic enhancement and biological loss of function we demonstrate conclusively that VEP measures myelin status and is thereby a validated tool for preclinical verification of remyelination.

Subclinical retinal injury occurs in pediatric-onset MS patients without a history of ON. As in adult-onset MS, substantial GCL thinning is present in eyes with prior ON. Finally, greater retinal axonal injury occurs in boys compared to girls.

We read with great interest the paper by Sotirchos et al. 1 in the June issue of Annals of Neurology describing retinal atrophy in multiple sclerosis (MS). Although we found their results generally consistent with data from our deeply phenotyped observational cohort (UCSF EPIC), 2 their described pattern of inner nuclear layer (INL) change differs significantly from our own findings. Sotirchos reported that increasing age is associated with increasingly rapid INL thinning, a finding most prominent in patients with progressive MS (PMS). This suggests that at every age patients are losing INL thickness, which therefore