The role of microglia in degenerative and regenerative processes after damage of the nervous system remains ambiguous, partially due to the paucity of appropriate investigative methods. Here, we show that treatment with the pharmacological colony stimulating factor 1 receptor inhibitor PLX5622 specifically eliminated microglia in murine retinae and optic nerves with high efficiency. Interestingly, time course and extent of retinal ganglion cell (RGC) degeneration after optic nerve crush remained unaffected upon microglia depletion, although remnants of prelabeled apoptotic RGCs were not cleared from the retina in these animals. In addition, microglia depletion neither affected the induction of regeneration associated genes upon optic nerve injury nor the increased regenerative potential of RGCs upon lens injury (LI). However, although the repopulation of the optic nerve lesion site by astrocytes was significantly delayed upon microglia depletion, spontaneous and LI-induced axon regeneration were unaffected by PLX5622 treatment or peripheral macrophage depletion by clodronate liposome treatment. Only concurrent double depletion of microglia and infiltrated macrophages slightly, but significantly, compromised optic nerve regeneration. Therefore, microglia are not essentially involved in RGC degeneration or axonal regeneration after acute CNS injury.

Implications of GSK3 activity for axon regeneration are often inconsistent, if not controversial. Sustained GSK3 activity in GSK3 knock-in mice reportedly accelerates peripheral nerve regeneration via increased MAP1B phosphorylation and concomitantly reduces microtubule detyrosination. In contrast, the current study shows that lens injury-stimulated optic nerve regeneration was significantly compromised in these knock-in mice. Phosphorylation of MAP1B and CRMP2 was expectedly increased in retinal ganglion cell (RGC) axons upon enhanced GSK3 activity, but, surprisingly, no GSK3-mediated CRMP2 inhibition was detected in sciatic nerves, thus revealing a fundamental difference between central and peripheral axons. Conversely, genetic or shRNA-mediated conditional KO/knockdown of GSK3β reduced inhibitory phosphorylation of CRMP2 in RGCs and improved optic nerve regeneration. Accordingly, GSK3β KO-mediated neurite growth promotion and myelin disinhibition were abrogated by CRMP2 inhibition and largely mimicked in WT neurons upon expression of constitutively active CRMP2 (CRMP2). These results underscore the prevalent requirement of active CRMP2 for optic nerve regeneration. Strikingly, expression of CRMP2 in GSK3 RGCs further boosted optic nerve regeneration, with axons reaching the optic chiasm within 3 wk. Thus, active GSK3 can also markedly promote axonal growth in central nerves if CRMP2 concurrently remains active. Similar to peripheral nerves, GSK3-mediated MAP1B phosphorylation/activation and the reduction of microtubule detyrosination contributed to this effect. Overall, these findings reconcile conflicting data on GSK3-mediated axon regeneration. In addition, the concept of complementary modulation of normally antagonistically targeted GSK3 substrates offers a therapeutically applicable approach to potentiate the regenerative outcome in the injured CNS.

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)

BackgroundIn multiple sclerosis (MS), neurodegeneration is the main reason for chronic disability. Alpha-lipoic acid (LA) is a naturally occurring antioxidant which has recently been demonstrated to reduce the rate of brain atrophy in progressive MS. However, it remains uncertain if it is also beneficial in the early, more inflammatory-driven phases. As clinical studies are costly and time consuming, optic neuritis (ON) is often used for investigating neuroprotective or regenerative therapeutics. We aimed to investigate the prospect for success of a clinical ON trial using an experimental autoimmune encephalomyelitis-optic neuritis (EAE-ON) model with visual system readouts adaptable to a clinical ON trial.MethodsUsing an in vitro cell culture model for endogenous oxidative stress, we compared the neuroprotective capacity of racemic LA with the R/S-enantiomers and its reduced form. In vivo, we analyzed retinal neurodegeneration using optical coherence tomography (OCT) and the visual function by optokinetic response (OKR) in MOG35–55-induced EAE-ON in C57BL/6J mice. Ganglion cell counts, inflammation, and demyelination were assessed by immunohistological staining of retinae and optic nerves.ResultsAll forms of LA provided equal neuroprotective capacities in vitro. In EAE-ON, prophylactic LA therapy attenuated the clinical EAE score and prevented the thinning of the inner retinal layer while therapeutic treatment was not protective on visual outcomes.ConclusionsA prophylactic LA treatment is necessary to protect from visual loss and retinal thinning in EAE-ON, suggesting that a clinical ON trial starting therapy after the onset of symptoms may not be successful.Electronic supplementary materialThe online version of this article (10.1186/s12974-018-1111-y) contains supplementary material, which is available to authorized users.

Knockout of phosphatase and tensin homolog (PTEN −/− ) is neuroprotective and promotes axon regeneration in mature neurons. Elevation of mTOR activity in injured neurons has been proposed as the primary underlying mechanism. Here we demonstrate that PTEN −/− also abrogates the inhibitory activity of GSK3 on collapsin response mediator protein 2 (CRMP2) in retinal ganglion cell (RGC) axons. Moreover, maintenance of GSK3 activity in Gsk3 S/A knockin mice significantly compromised PTEN −/− -mediated optic nerve regeneration as well as the activity of CRMP2, and to a lesser extent, mTOR. These GSK3 S/A mediated negative effects on regeneration were rescued by viral expression of constitutively active CRMP2 T/A , despite decreased mTOR activation. Gsk3 S/A knockin or CRMP2 inhibition also decreased PTEN −/− mediated neurite growth of RGCs in culture and disinhibition towards CNS myelin. Thus, the GSK3/CRMP2 pathway is essential for PTEN −/− mediated axon regeneration. These new mechanistic insights may help to find novel strategies to promote axon regeneration.

Highlights d MLP is a regeneration-associated gene expressed in adult neurons of mammals d As an actin cross-linker, MLP facilitates filopodia formation in axonal growth cones d MLP overexpression facilitates CNS and PNS axon regeneration

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.