Noninvasive Light Flicker Stimulation Promotes Optic Nerve Regeneration by Activating Microglia and Enhancing Neural Plasticity in Zebrafish.

Purpose: Forty-hertz light flicker stimulation has been proven to reduce neurodegeneration, but its effect on optic nerve regeneration is unclear. This study explores the effect of 40-Hz light flicker in promoting optic nerve regeneration in zebrafish and investigates the underlying mechanisms. Methods: Wild-type and mpeg1:EGFP zebrafish were used to establish a model of optic nerve crush. Biocytin tracing and hematoxylin and eosin staining were employed to observe whether 40-Hz light flicker promotes regeneration of retinal ganglion cell axons and dendrites. Optomotor and optokinetic responses were evaluated to assess recovery of visual function. Immunofluorescence staining of mpeg1:EGFP zebrafish was performed to observe changes in microglia. Differentially expressed genes that promote optic nerve regeneration following 40-Hz light flicker stimulation were identified and validated through RNA-sequencing analysis and quantitative real-time PCR (qRT-PCR). Results: Zebrafish exhibited spontaneous optic nerve regeneration after optic nerve injury and restored visual function. We observed that 40-Hz light flicker significantly activated microglia following optic nerve injury and promoted regeneration of retinal ganglion cell axons and dendrites, as well as recovery of visual function. Transcriptomics and qRT-PCR analyses revealed that 40-Hz light flicker increased the expression of genes associated with neuronal plasticity, including bdnf, npas4a, fosab, fosb, egr4, and ier2a. Conclusions: To our knowledge, this study is the first to demonstrate that 40-Hz light flicker stimulation promotes regeneration of retinal ganglion cell axons and dendrites and recovery of visual function in zebrafish, which is associated with microglial activation and enhancement of neural plasticity.

Large-scale in-silico analysis of CSF dynamics within the subarachnoid space of the optic nerve.

BACKGROUND: Impaired cerebrospinal fluid (CSF) dynamics is involved in the pathophysiology of neurodegenerative diseases of the central nervous system and the optic nerve (ON), including Alzheimer's and Parkinson's disease, as well as frontotemporal dementia. The smallness and intricate architecture of the optic nerve subarachnoid space (ONSAS) hamper accurate measurements of CSF dynamics in this space, and effects of geometrical changes due to pathophysiological processes remain unclear. The aim of this study is to investigate CSF dynamics and its response to structural alterations of the ONSAS, from first principles, with supercomputers. METHODS: Large-scale in-silico investigations were performed by means of computational fluid dynamics (CFD) analysis. High-order direct numerical simulations (DNS) have been carried out on ONSAS geometry at a resolution of 1.625 µm/pixel. Morphological changes on the ONSAS microstructure have been examined in relation to CSF pressure gradient (CSFPG) and wall strain rate, a quantitative proxy for mass transfer of solutes. RESULTS: A physiological flow speed of 0.5 mm/s is achieved by imposing a hydrostatic pressure gradient of 0.37-0.67 Pa/mm across the ONSAS structure. At constant volumetric rate, the relationship between pressure gradient and CSF-accessible volume is well captured by an exponential curve. The ONSAS microstructure exhibits superior mass transfer compared to other geometrical shapes considered. An ONSAS featuring no microstructure displays a threefold smaller surface area, and a 17-fold decrease in mass transfer rate. Moreover, ONSAS trabeculae seem key players in mass transfer. CONCLUSIONS: The present analysis suggests that a pressure drop of 0.1-0.2 mmHg over 4 cm is sufficient to steadily drive CSF through the entire subarachnoid space. Despite low hydraulic resistance, great heterogeneity in flow speeds puts certain areas of the ONSAS at risk of stagnation. Alterations of the ONSAS architecture aimed at mimicking pathological conditions highlight direct relationships between CSF volume and drainage capability. Compared to the morphological manipulations considered herein, the original ONSAS architecture seems optimized towards providing maximum mass transfer across a wide range of pressure gradients and volumetric rates, with emphasis on trabecular structures. This might shed light on pathophysiological processes leading to damage associated with insufficient CSF flow in patients with optic nerve compartment syndrome.

Optic nerve regeneration: Potential treatment approaches.

The optic nerve, predominantly constituted by the axons of retinal ganglion cells (RGCs), lacks the ability to regenerate and re-establish function after injury. RGCs are crucial for visual function, and thus, RGC death contributes to the development of numerous progressive neurodegenerative optic neuropathies including glaucoma, ischemic optic neuropathy, and optic neuritis. Regenerating optic nerve axons poses numerous challenges due to factors such as the intricate and inhibitory conditions that exist within their environment, intrinsic breaks to regeneration, and the geometric tortuosity that offers physical hindrance to axon growth. However, recent research advancements offer hope for clinically meaningful regeneration for those who suffer from optic nerve damage. In this review, we highlight the current treatment approaches for optic nerve axon regeneration.

A Comparative Investigation of Axon-Blood Vessel Growth Interaction in the Regenerating Sciatic and Optic Nerves in Adult Mice.

The vascular and the nervous systems share similarities in addition to their complex role in providing oxygen and nutrients to all cells. Both are highly branched networks that frequently grow close to one another during development. Vascular patterning and neural wiring share families of guidance cues and receptors. Most recently, this relationship has been investigated in terms of peripheral nervous system (PNS) regeneration, where nerves and blood vessels often run in parallel so endothelial cells guide the formation of the Büngner bands which support axonal regeneration. Here, we characterized the vascular response in regenerative models of the central and peripheral nervous system. After sciatic nerve crush, followed by axon regeneration, there was a significant increase in the blood vessel density 7 days after injury. In addition, the optic nerve crush model was used to evaluate intrinsic regenerative potential activated with a combined treatment that stimulated retinal ganglion cells (RGCs) regrowth. We observed that a 2-fold change in the total number of blood vessels occurred 7 days after optic nerve crush compared to the uncrushed nerve. The difference increased up to a 2.7-fold change 2 weeks after the crush. Interestingly, we did not observe differences in the total number of blood vessels 2 weeks after crush, compared to animals that had received combined treatment for regeneration and controls. Therefore, the vascular characterization showed that the increase in vascular density was not related to the efficiency of both peripheral and central axonal regeneration.

Inflammatory Mediators of Axon Regeneration in the Central and Peripheral Nervous Systems.

Although most pathways in the mature central nervous system cannot regenerate when injured, research beginning in the late 20th century has led to discoveries that may help reverse this situation. Here, we highlight research in recent years from our laboratory identifying oncomodulin (Ocm), stromal cell-derived factor (SDF)-1, and chemokine CCL5 as growth factors expressed by cells of the innate immune system that promote axon regeneration in the injured optic nerve and elsewhere in the central and peripheral nervous systems. We also review the role of ArmC10, a newly discovered Ocm receptor, in mediating many of these effects, and the synergy between inflammation-derived growth factors and complementary strategies to promote regeneration, including deleting genes encoding cell-intrinsic suppressors of axon growth, manipulating transcription factors that suppress or promote the expression of growth-related genes, and manipulating cell-extrinsic suppressors of axon growth. In some cases, combinatorial strategies have led to unprecedented levels of nerve regeneration. The identification of some similar mechanisms in human neurons offers hope that key discoveries made in animal models may eventually lead to treatments to improve outcomes after neurological damage in patients.

Effects of head-down tilt on optic nerve sheath diameter in healthy subjects.

PURPOSE: Intracranial pressure increases in head-down tilt (HDT) body posture. This study evaluated the effect of HDT on the optic nerve sheath diameter (ONSD) in normal subjects. METHODS: Twenty six healthy adults (age 28 [4.7] years) participated in seated and 6° HDT visits. For each visit, subjects presented at 11:00 h for baseline seated scans and then maintained a seated or 6° HDT posture from 12:00 to 15:00 h. Three horizontal axial and three vertical axial scans were obtained at 11:00, 12:00 and 15:00 h with a 10 MHz ultrasonography probe on the same eye, randomly chosen per subject. At each time point, horizontal and vertical ONSD (mm) were quantified by averaging three measures taken 3 mm behind the globe. RESULTS: In the seated visit, ONSDs were similar across time (p > 0.05), with an overall mean (standard deviation) of 4.71 (0.48) horizontally and 5.08 (0.44) vertically. ONSD was larger vertically than horizontally at each time point (p < 0.001). In the HDT visit, ONSD was significantly enlarged from baseline at 12:00 and 15:00 h (p < 0.001 horizontal and p < 0.05 vertical). Mean (standard error) horizontal ONSD change from baseline was 0.37 (0.07) HDT versus 0.10 (0.05) seated at 12:00 h (p = 0.002) and 0.41 (0.09) HDT versus 0.12 (0.06) seated at 15:00 h (p = 0.002); mean vertical ONSD change was 0.14 (0.07) HDT versus -0.07 (0.04) seated at 12:00 h (p = 0.02) and 0.19 (0.06) HDT versus -0.03 (0.04) seated at 15:00 h (p = 0.01). ONSD change in HDT was similar between 12:00 and 15:00 h (p ≥ 0.30). Changes at 12:00 h correlated with those at 15:00 h for horizontal (r = 0.78, p < 0.001) and vertical ONSD (r = 0.73, p < 0.001). CONCLUSION: The ONSD increased when body posture transitioned from seated to HDT position without any further change at the end of the 3 h in HDT.

The Optic Nerve Crush Injury Paradigm in African Turquoise Killifish to Study Axonal Regeneration in an Aged Environment.

In our graying world population, we are increasingly facing brain injuries and age-associated neurodegenerative diseases, which are often characterized by axonal pathology. Here, we propose the killifish visual/retinotectal system as a model for investigating central nervous system repair, more specifically axonal regeneration, in an aging context. We first describe an optic nerve crush (ONC) injury paradigm in killifish to induce and study both de- and regeneration of retinal ganglion cells (RGCs) and their axons. Subsequently, we summarize several methods for mapping different steps of the regenerative process-namely, axonal regrowth and synapse reformation-using retro- and anterograde tracing methods, (immuno)histochemistry, and morphometrical analyses.

Secondary Degeneration Impairs Myelin Ultrastructural Development in Adulthood following Adolescent Neurotrauma in the Rat Optic Nerve.

Adolescence is a critical period of postnatal development characterized by social, emotional, and cognitive changes. These changes are increasingly understood to depend on white matter development. White matter is highly vulnerable to the effects of injury, including secondary degeneration in regions adjacent to the primary injury site which alters the myelin ultrastructure. However, the impact of such alterations on adolescent white matter maturation is yet to be investigated. To address this, female piebald-virol-glaxo rats underwent partial transection of the optic nerve during early adolescence (postnatal day (PND) 56) with tissue collection two weeks (PND 70) or three months later (PND 140). Axons and myelin in the transmission electron micrographs of tissue adjacent to the injury were classified and measured based on the appearance of the myelin laminae. Injury in adolescence impaired the myelin structure in adulthood, resulting in a lower percentage of axons with compact myelin and a higher percentage of axons with severe myelin decompaction. Myelin thickness did not increase as expected into adulthood after injury and the relationship between the axon diameter and myelin thickness in adulthood was altered. Notably, dysmyelination was not observed 2 weeks postinjury. In conclusion, injury in adolescence altered the developmental trajectory, resulting in impaired myelin maturation when assessed at the ultrastructural level in adulthood.

Finite element modeling of effects of tissue property variation on human optic nerve tethering during adduction.

Tractional tethering by the optic nerve (ON) on the eye as it rotates towards the midline in adduction is a significant ocular mechanical load and has been suggested as a cause of ON damage induced by repetitive eye movements. We designed an ocular finite element model (FEM) simulating 6° incremental adduction beyond the initial configuration of 26° adduction that is the observed threshold for ON tethering. This FEM permitted sensitivity analysis of ON tethering using observed material property variations in measured hyperelasticity of the anterior, equatorial, posterior, and peripapillary sclera; and the ON and its sheath. The FEM predicted that adduction beyond the initiation of ON tethering concentrates stress and strain on the temporal side of the optic disc and peripapillary sclera, the ON sheath junction with the sclera, and retrolaminar ON neural tissue. However, some unfavorable combinations of tissue properties within the published ranges imposed higher stresses in these regions. With the least favorable combinations of tissue properties, adduction tethering was predicted to stress the ON junction and peripapillary sclera more than extreme conditions of intraocular and intracranial pressure. These simulations support the concept that ON tethering in adduction could induce mechanical stresses that might contribute to ON damage.

Epigenetic Regulation of Optic Nerve Development, Protection, and Repair.

Epigenetic factors are known to influence tissue development, functionality, and their response to pathophysiology. This review will focus on different types of epigenetic regulators and their associated molecular apparatus that affect the optic nerve. A comprehensive understanding of epigenetic regulation in optic nerve development and homeostasis will help us unravel novel molecular pathways and pave the way to design blueprints for effective therapeutics to address optic nerve protection, repair, and regeneration.

Neuronal energy use and brain evolution.

Consider how advantageous it might be to have eyes on our hands, rather than on our faces: depth perception would be improved by the greater distance between the eyes, and it would be easy to look into relatively inaccessible spaces by appropriate movement of the hands. The absence of mammals that use this visual strategy draws attention to constraints on how evolution is able to 'design' the nervous system. Energy use in particular, in this case the large amount of energy that would be needed to send visual information along the ∼106 optic nerve axons over the length of the arms to the brain (instead of along the much shorter optic nerve), imposes significant design constraints on the nervous system.

[Regeneration of the optic nerve-Will it one day be reality?] / Regeneration des Sehnerven ­ Wird das einmal Realität?

BACKGROUND: Adult mammalian and human neurons of the central nervous system (CNS) lack the ability to spontaneously regenerate damaged axons. This dilemma of many CNS diseases is still an unsolved problem. OBJECTIVE: The purpose of this article is to examine the question which options have been investigated in more detail in recent years and offer approaches. METHODS: A web-based search of all articles published between 1958 to the present regarding regeneration of retinal ganglion cells was carried out. RESULTS: Over the last three decades it has been shown that axonal regeneration is possible under certain conditions when intrinsic and extrinsic factors are manipulated in retinal ganglion cells and in the optic nerve. Although there is still a long way to go, experimental regenerative approaches are already visible; however, it will take several years or decades before these can be approximately implemented in practice.

[Research advances in classifications and functions of retinal ganglion cells].

Retinal ganglion cells (RGCs) are the most important type of neurons in the visual pathway. RGC axons exit the eye to form the optic nerve, which connects with the brain. The visual signals carried by RGC axons establish the only link between the outside world and our internal perception of sight. Researches on the morphological, physiological, molecular, and mosaic features of RGCs are of great significance. This article reviews the research advances of RGC classifications, definitive types of RGCs, and selective vulnerability of specific RGC types after various injuries.

Three-dimensional multilayer concentric bipolar electrodes restrict spatial activation in optic nerve stimulation.

Objective.Intraneural nerve interfaces often operate in a monopolar configuration with a common and distant ground electrode. This configuration leads to a wide spreading of the electric field. Therefore, this approach is suboptimal for intraneural nerve interfaces when selective stimulation is required.Approach.We designed a multilayer electrode array embedding three-dimensional concentric bipolar (CB) electrodes. First, we validated the higher stimulation selectivity of this new electrode array compared to classical monopolar stimulation using simulations. Next, we compared themin-vivoby intraneural stimulation of the rabbit optic nerve and recording evoked potentials in the primary visual cortex.Main results.Simulations showed that three-dimensional CB electrodes provide a high localisation of the electric field in the tissue so that electrodes are electrically independent even for high electrode density. Experimentsin-vivohighlighted that this configuration restricts spatial activation in the visual cortex due to the fewer fibres activated by the electric stimulus in the nerve.Significance.Highly focused electric stimulation is crucial to achieving high selectivity in fibre activation. The multilayer array embedding three-dimensional CB electrodes improves selectivity in optic nerve stimulation. This approach is suitable for other neural applications, including bioelectronic medicine.

White matter tract conductivity is resistant to wide variations in paranodal structure and myelin thickness accompanying the loss of Tyro3: an experimental and simulated analysis.

Myelination within the central nervous system (CNS) is crucial for the conduction of action potentials by neurons. Variation in compact myelin morphology and the structure of the paranode are hypothesised to have significant impact on the speed of action potentials. There are, however, limited experimental data investigating the impact of changes in myelin structure upon conductivity in the central nervous system. We have used a genetic model in which myelin thickness is reduced to investigate the effect of myelin alterations upon action potential velocity. A detailed examination of the myelin ultrastructure of mice in which the receptor tyrosine kinase Tyro3 has been deleted showed that, in addition to thinner myelin, these mice have significantly disrupted paranodes. Despite these alterations to myelin and paranodal structure, we did not identify a reduction in conductivity in either the corpus callosum or the optic nerve. Exploration of these results using a mathematical model of neuronal conductivity predicts that the absence of Tyro3 would lead to reduced conductivity in single fibres, but would not affect the compound action potential of multiple myelinated neurons as seen in neuronal tracts. Our data highlight the importance of experimental assessment of conductivity and suggests that simple assessment of structural changes to myelin is a poor predictor of neural functional outcomes.

Imaging Axonal Transport in Ex Vivo Central and Peripheral Nerves.

Neurones are highly polarized cells with extensive axonal projections that rely on transport of proteins, RNAs, and organelles in a bidirectional manner to remain healthy. This process, known as axonal transport, can be imaged in real time through epifluorescent imaging of fluorescently labeled proteins, organelles, and other cargoes. While this is most conveniently done in primary neuronal cultures, it is more physiologically relevant when carried out in the context of a developed nerve containing both axons and glia. Here we outline how to image axonal transport ex vivo in sciatic and optic nerves, and the fimbria of the fornix. These methods could be altered to image other fluorescently labeled molecules, as well as different mechanisms of intracellular transport.

Ouabain-Na+/K+-ATPase Signaling Regulates Retinal Neuroinflammation and ROS Production Preventing Neuronal Death by an Autophagy-Dependent Mechanism Following Optic Nerve Axotomy In Vitro.

Ouabain is a classic Na+K+ATPase ligand and it has been described to have neuroprotective effects on neurons and glial cells at nanomolar concentrations. In the present work, the neuroprotective and immunomodulatory potential of ouabain was evaluated in neonatal rat retinal cells using an optic nerve axotomy model in vitro. After axotomy, cultured retinal cells were treated with ouabain (3 nM) at different periods. The levels of important inflammatory receptors in the retina such as TNFR1/2, TLR4, and CD14 were analyzed. We observed that TNFR1, TLR4, and CD14 were decreased in all tested periods (15 min, 45 min, 24 h, and 48 h). On the other hand, TNFR2 was increased after 24 h, suggesting an anti-inflammatory potential for ouabain. Moreover, we showed that ouabain also decreased Iba-1 (microglial marker) density. Subsequently, analyses of retrograde labeling of retinal ganglion cells (RGC) were performed after 48 h and showed that ouabain-induced RGC survival depends on autophagy. Using an autophagy inhibitor (3-methyladenine), we observed a complete blockage of the ouabain effect. Western blot analyses showed that ouabain increases the levels of autophagy proteins (LC3 and Beclin-1) coupled to p-CREB transcription factor and leads to autophagosome formation. Additionally, we found that the ratio of cleaved/pro-caspase-3 did not change after ouabain treatment; however, p-JNK density was enhanced. Also, ouabain decreased reactive oxygen species production immediately after axotomy. Taken together, our results suggest that ouabain controls neuroinflammation in the retina following optic nerve axotomy and promotes RGC neuroprotection through activation of the autophagy pathway.

The Future of Stem Cells and Their Derivates in the Treatment of Glaucoma. A Critical Point of View.

This review focuses on the clinical translation of preclinical studies, especially those that have used stem cells in the treatment of glaucoma, with an emphasis on optic nerve regeneration. The studies referred to in the review aim to treat optic nerve atrophy, while cell therapies targeting other sites in the eye, such as the trabecular meshwork, have not been addressed. Such complex and varied pathophysiological mechanisms that lead to glaucoma may explain the fact that although stem cells have a high capacity of neuronal regeneration, the treatments performed did not have the expected results and the promise offered by animal studies was not achieved. By analyzing the facts associated with failure, important lessons are to be learned: the type of stem cells that are used, the route of administration, the selection of patients eligible for these treatments, additional therapies that support stem cells transplantation and their mode of action, methods of avoiding the host's immune response. Many of these problems could be solved using exosomes (EV), but also miRNA, which allows more targeted approaches with minimal side effects.