Retinal Axons Research Articles

Torsion-Induced Traumatic Optic Neuropathy (TITON): A Physiologically Relevant Animal Model of Traumatic Optic Neuropathy.

Traumatic optic neuropathy (TON) is a common cause of irreversible blindness following head injury. TON is characterized by axon damage in the optic nerve followed by retinal ganglion cell death in the days and weeks following injury. At present, no therapeutic or surgical approach has been found to offer any benefit beyond observation alone. This is due in part to the lack of translational animal models suitable for understanding mechanisms and evaluating candidate treatments. In this study, we developed a rat model of TON in which the eye is rapidly rotated, inflicting mechanical stress on the optic nerve and leading to significant visual deficits. These functional deficits were thoroughly characterized up to one week after injury using electrophysiology and immunohistochemistry. The photopic negative response (PhNR) of the light adapted full field electroretinogram (LA ffERG) was significantly altered following injury. This correlated with increased biomarkers of retinal stress, axon disruption, and ganglion cell death. Together, this evidence suggests the utility of our model for mimicking clinically relevant TON and that the PhNR may be an early diagnostic for TON. We also found indirect evidence that ketamine, which was used for anesthesia, may ameliorate TON. Future studies will utilize this animal model for evaluation of candidate treatments.

Axon guidance during mouse central nervous system regeneration is required for specific brain innervation.

Reconstructing functional neuronal circuits is one major challenge of central nervous system repair. Through activation of pro-growth signaling pathways, some neurons achieve long-distance axon regrowth. Yet, functional reconnection has hardly been obtained, as these regenerating axons fail to resume their initial trajectory and reinnervate their proper target. Axon guidance is considered to be active only during development. Here, using the mouse visual system, we show that axon guidance is still active in the adult brain in regenerative conditions. We highlight that regenerating retinal ganglion cell axons avoid one of their primary targets, the suprachiasmatic nucleus (SCN), due to Slit/Robo repulsive signaling. Together with promoting regeneration, silencing Slit/Robo invivo enables regenerating axons to enter the SCN and form active synapses. The newly formed circuit is associated with neuronal activation and functional recovery. Our results provide evidence that axon guidance mechanisms are required to reconnect regenerating axons to specific brain nuclei.

Mistargeted retinal axons form synaptically segregated subcircuits in the visual thalamus of albino mice.

A Hebbian model of circuit remodeling predicts that two sets of inputs with sufficiently distinct activity patterns will synaptically capture separate sets of target cells. Mice in which a subset of retinal ganglion cells (RGCs) target the wrong region of the dorsal lateral geniculate nucleus (dLGN) provide the conditions for testing this prediction. In albino mice, mistargeted RGC axons form an island of terminals that is distinct from the surrounding neuropil. Blocking retinal activity during development prevents the formation of this island. However, the synaptic connectivity of the island was unknown. Here, we combine light and electron microscopy to determine if this activity-dependent island of axon terminals represent a synaptically segregated subcircuit. We reconstructed the microcircuitry of the boundary between the island and non-island RGCs and found a remarkably strong segregation within retinogeniculate connectivity. We conclude that, when sets of retinal input are established in the wrong part of the dLGN, the developing circuitry responds by forming a synaptically isolated subcircuit from the otherwise fully connected network. The fact that there is a developmental starting condition that can induce a synaptically segregated microcircuit has important implications for our understanding of the organization of visual circuits and for our understanding of the implementation of activity dependent development.

Complement Factor B Inhibition or Deletion Is Not Sufficient to Prevent Neurodegeneration in a Murine Model of Glaucoma.

Purpose: Activation of the classical complement pathway is thought to contribute to the development and progression of glaucoma. The role of alternative complement or amplification pathways in glaucoma is not well understood. We evaluated complement factor B (FB) expression in postmortem human ocular tissues with or without glaucoma and the effect of FB inhibition and deletion in a mouse ocular hypertensive model of glaucoma induced by photopolymerized hyaluronic acid glycidyl methacrylate (HAGM). Methods: Human CFB mRNA in human eyes was assessed by RNAscope and TaqMan. HAGM model was performed on C57BL6/J mice. The effect of FB in HAGM model was evaluated with an oral FB inhibitor and Cfb-/- mice. Complement mRNA and proteins in mouse eyes were assessed by TaqMan and western blot, respectively. Results: CFB mRNA in human glaucomatous macular neural retina and optic nerve head was upregulated. Cfb mRNA is also upregulated in the HAGM model. Oral FB inhibitor, ED-79-GX17, dosed daily at 200 mg/kg for 3 days after intraocular pressure (IOP) induction in wild-type mice showed complement inhibition in ocular tissues and significantly inhibited systemic complement levels. Daily dosing of ED-79-GX17 for 30 days or Cfb deletion was also unable to prevent retinal ganglion cell or axon loss 30 days after IOP induction in mice. Conclusion: The alternative complement component FB may not substantially contribute to RGC loss in the HAGM mouse glaucoma model despite upregulation of Cfb expression and activation of the alternative pathway. The relevance of these findings to human glaucoma remains to be determined.

Transcriptional profiling of retinal astrocytes identifies a specific marker and points to functional specialization.

Astrocyte heterogeneity is an increasingly prominent research topic, and studies in the brain have demonstrated substantial variation in astrocyte form and function, both between and within regions. In contrast, retinal astrocytes are not well understood and remain incompletely characterized. Along with optic nerve astrocytes, they are responsible for supporting retinal ganglion cell axons and an improved understanding of their role is required. We have used a combination of microdissection and Ribotag immunoprecipitation to isolate ribosome-associated mRNA from retinal astrocytes and investigate their transcriptome, which we also compared to astrocyte populations in the optic nerve. Astrocytes from these regions are transcriptionally distinct, and we identified retina-specific astrocyte genes and pathways. Moreover, although they share much of the "classical" gene expression patterns of astrocytes, we uncovered unexpected variation, including in genes related to core astrocyte functions. We additionally identified the transcription factor Pax8 as a highly specific marker of retinal astrocytes and demonstrated that these astrocytes populate not only the retinal surface, but also the prelaminar region at the optic nerve head. These findings are likely to contribute to a revised understanding of the role of astrocytes in the retina.

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

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. 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). 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. 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.

Mesenchymal stem cells for repairing glaucomatous optic nerve.

Glaucoma is a common and complex neurodegenerative disease characterized by progressive loss of retinal ganglion cells (RGCs) and axons. Currently, there is no effective method to address the cause of RGCs degeneration. However, studies on neuroprotective strategies for optic neuropathy have increased in recent years. Cell replacement and neuroprotection are major strategies for treating glaucoma and optic neuropathy. Regenerative medicine research into the repair of optic nerve damage using stem cells has received considerable attention. Stem cells possess the potential for multidirectional differentiation abilities and are capable of producing RGC-friendly microenvironments through paracrine effects. This article reviews a thorough researches of recent advances and approaches in stem cell repair of optic nerve injury, raising the controversies and unresolved issues surrounding the future of stem cells.

Plexin B3 guides axons to cross the midline in vivo.

During the development of neural circuits, axons are guided by a variety of molecular cues to navigate through the brain and establish precise connections with correct partners at the right time and place. Many axon guidance cues have been identified and they play pleiotropic roles in not only axon guidance but also axon fasciculation, axon pruning, and synaptogenesis as well as cell migration, angiogenesis, and bone formation. In search of receptors for Sema3E in axon guidance, we unexpectedly found that Plexin B3 is highly expressed in retinal ganglion cells of zebrafish embryos when retinal axons are crossing the midline to form the chiasm. Plexin B3 has been characterized to be related to neurodevelopmental disorders. However, the investigation of its pathological mechanisms is hampered by the lack of appropriate animal model. We provide evidence that Plexin B3 is critical for axon guidance in vivo. Plexin B3 might function as a receptor for Sema3E while Neuropilin1 could be a co-receptor. The intracellular domain of Plexin B3 is required for Semaphorin signaling transduction. Our data suggest that zebrafish could be an ideal animal model for investigating the role and mechanisms of Sema3E and Plexin B3 in vivo.

Investigation of the Effects of a Novel NOX2 Inhibitor, GLX7013170, against Glutamate Excitotoxicity and Diabetes Insults in the Retina.

Glutamate excitotoxicity and oxidative stress represent two major pathological mechanisms implicated in retinal disorders. In Diabetic Retinopathy (DR), oxidative stress is correlated to NADPH oxidase (NOX), a major source of Reactive Oxygen Species (ROS), and glutamate metabolism impairments. This study investigated the role of NOX2 and the novel NOX2 inhibitor, GLX7013170, in two models of a) retinal AMPA excitotoxicity [AMPA+GLX7013170 (10-4 M, intravitreally)] and b) early-stage DR paradigm (ESDR), GLX7013170: 14-day therapeutic treatment (topically, 20 μL/eye, 10 mg/mL (300 × 10-4 M), once daily) post-streptozotocin (STZ)-induced DR. Immunohistochemical studies for neuronal markers, nitrotyrosine, micro/macroglia, and real-time PCR, Western blot, and glutamate colorimetric assays were conducted. Diabetes increased NOX2 expression in the retina. NOX2 inhibition limited the loss of NOS-positive amacrine cells and the overactivation of micro/macroglia in both models. In the diabetic retina, GLX7013170 had no effect on retinal ganglion cell axons, but reduced oxidative damage, increased Bcl-2, reduced glutamate levels, and partially restored excitatory amino acid transporter (EAAT1) expression. These results suggest that NOX2 in diabetes is part of the triad, oxidative stress, NOX, and glutamate excitotoxicity, key players in the induction of DR. GLX7013170 is efficacious as a neuroprotective/anti-inflammatory agent and a potential therapeutic in retinal diseases, including ESDR.

The Optic Nerve at Stake: Update on Environmental Factors Modulating Expression of Leber's Hereditary Optic Neuropathy.

Optic neuropathies are characterized by the degeneration of the optic nerves and represent a considerable individual and societal burden. Notably, Leber's hereditary optic neuropathy (LHON) is a devastating vision disease caused by mitochondrial gene mutations that hinder oxidative phosphorylation and increase oxidative stress, leading to the loss of retinal ganglion neurons and axons. Loss of vision is rapid and severe, predominantly in young adults. Penetrance is incomplete, and the time of onset is unpredictable. Recent findings revealed that the incidence of genetic LHON susceptibility is around 1 in 1000, much higher than believed till now. Environmental factors are critical in LHON triggering or severity. Families at risk have a very strong demand for how to prevent the onset or limit the severity of the disease. Here, we review recent knowledge of the extrinsic determinants of LHON expression, including lifestyle, dietary supplements, common chemicals, and drugs.

Distribution, development, and identity of retinal ganglion cells labeled in the Sert-Cre reporter mouse.

The mouse retina contains over 40 types of retinal ganglion cells (RGCs) that differ in morphology, function, or gene expression. RGCs also differ by whether their axons target the brain.s ipsilateral or contralateral hemisphere. Contralaterally projecting RGCs (contraRGCs) are widespread in mouse retina, whereas ipsilateral projecting RGCs (ipsiRGCs) are confined to the ventro-temporal (VT) crescent of retina. In this study, we employed the Sert-Cre transgenic line, which had been reported to selectively label ipsiRGCs, to study ipsiRGCs during development. Although the number of Cre-expressing ipsiRGCs did not significantly increase with postnatal age, the region of retina that they occupied did, and by adulthood represented ~30% of the retinal surface. Unexpectedly, genetic ablation of Sert-Cre cells failed to fully disrupt ipsilateral projecting retinal axons, suggesting that not all ipsiRGCs generated Cre in Sert-Cre mice. To test this hypothesis, we retrogradely labeled ipsiRGCs in Sert-Cre mice which revealed that not all ipsiRGCs are labeled in Sert-Cre mice and a small population of contraRGCs flanking the VT crescent generates Cre in this line. These results do not negate the usefulness of the Sert-Cre mouse but do raise important caveats to the interpretation of such studies.

Retinal origin of orientation but not direction selective maps in the superior colliculus

Neurons in the mouse superior colliculus ("colliculus") are arranged in ordered spatial maps. While orientation-selective (OS) neurons form a concentric map aligned to the center of vision, direction-selective (DS) neurons are arranged in patches with changing preferences across the visual field. It remains unclear whether these maps are a consequence of feedforward input from the retina or local computations in the colliculus. To determine whether these maps originate in the retina, we mapped the local and global distribution of OS and DS retinal ganglion cell axon boutons using invivo two-photon calcium imaging. We found that OS boutons formed patches that matched the distribution of OS neurons within the colliculus. DS boutons displayed fewer regional specializations, better reflecting the organization of DS neurons in the retina. Both eyes convey similar orientation but different DS inputs to the colliculus, as shown in recordings from retinal explants. These data demonstrate that orientation and direction maps within the colliculus are independent, where orientation maps are likely inherited from the retina, but direction maps require additional computations.

In Vivo Imaging of Secondary Neurodegeneration Associated With Phosphatidylserine Externalization Along Axotomized Axons.

Axonal degeneration in acute and chronic disorders is well-characterized, comprising retrograde (proximal) and Wallerian (distal) degeneration, but the mechanism of propagation remains less understood. Laser injury with a diode-pumped solid-state 532nm laser was used to axotomize retinal ganglion cell axons. We used confocal in vivo imaging to demonstrate that phosphatidylserine externalization is a biomarker of early axonal degeneration after selective intraretinal axotomy. Quantitative dynamic analysis revealed that the rate of axonal degeneration was fastest within 40minutes, then decreased exponentially afterwards. Axonal degeneration was constrained within the same axotomized axonal bundles. Remarkably, axon degeneration arising from the site of injury induced a secondary degeneration of distal normal axons. Axonal degeneration in vivo is a progressive process associated with phosphatidylserine externalization, which can propagate not only along the axon but to adjacent uninjured axons. This finding has implications for acute and chronic neurodegenerative disorders associated with axonal injury.

Th1 cells contribute to retinal ganglion cell loss in glaucoma in a VCAM-1-dependent manner.

Glaucoma is a complex neurodegenerative disorder characterized by the progressive loss of retinal ganglion cells (RGC) and optic nerve axons, leading to irreversible visual impairment. Despite its clinical significance, the underlying mechanisms of glaucoma pathogenesis remain poorly understood. In this study, we aimed to unravel the multifaceted nature of glaucoma by investigating the interaction between T cells and retinas. By utilizing clinical samples, murine glaucoma models, and T cell transfer models, we made several key findings. Firstly, we observed that CD4+ T cells from glaucoma patients displayed enhanced activation and a bias towards T helper (Th) 1 responses, which correlated with visual impairment. Secondly, we identified the infiltration of Th1 cells into the retina, where they targeted RGC and integrated into the pro-inflammatory glial network, contributing to progressive RGC loss. Thirdly, we discovered that circulating Th1 cells upregulated vascular cell adhesion protein 1 (VCAM-1) on retinal microvessels, facilitating their entry into the neural retina. Lastly, we found that Th1 cells underwent functional reprogramming before reaching the retina, acquiring a phenotype associated with lymphocyte migration and neurodegenerative diseases. Our study provides novel insights into the role of peripheral CD4+ T cells in glaucoma pathogenesis, shedding light on the mechanisms underlying their infiltration into the retina and offering potential avenues for innovative therapeutic interventions in this sight-threatening disease.

Ciliary neurotrophic factor mediated growth of retinal ganglion cell axons on PGS/PCL scaffolds

Ciliary neurotrophic factor (CNTF) promotes survival and/or differentiation of a variety of neuronal cells including retinal ganglion cells (RGCs). Delivery of CNTF requires a suitable medium capable of mediating diffusion and premature release of CNTF within the target tissue. Polymeric tissue-engineered scaffolds have been readily used as substrates for cell transplantation, expansion, and differentiation and, as carriers of cell growth factors. Their functions to CNTF release for RGC proliferation have remained so far unexplored, especially to CNTF affinity to the scaffold and subsequent RGC fate. Electrospun poly(glycerol sebacate)/poly(ϵ-caprolactone) (PGS/PCL) biopolymer scaffolds have recently shown promising results in terms of supporting regeneration of RGC neurites. This work explores covalent immobilization of CNTF on PGS/PCL scaffold and the way immobilised CNTF mediates growth of RGC axons on the scaffold. An ex-vivo three-dimensional model of rodent optic nerve on PGS/PCL revealed that RGC explants cultured in CNTF mediated environment increased their neurite extensions after 20 d of cell culture employing neurite outgrowth measurements. The CNTF secretion on PGS/PCL scaffold was found bio-mimicking natural extracellular matrix of the cell target tissue and, consequently, has shown a potential to improve the overall efficacy of the RGC regeneration process.

CXCL12 promotes the crossing of retinal ganglion cell axons at the optic chiasm.

Binocular vision requires the segregation of retinal ganglion cell (RGC) axons extending from the retina into the ipsilateral and contralateral optic tracts. RGC axon segregation occurs at the optic chiasm, which forms at the ventral diencephalon midline. Using expression analyses, retinal explants and genetically modified mice, we demonstrate that CXCL12 (SDF1) is required for axon segregation at the optic chiasm. CXCL12 is expressed by the meninges bordering the optic pathway, and CXCR4 by both ipsilaterally and contralaterally projecting RGCs. CXCL12 or ventral diencephalon meninges potently promoted axon outgrowth from both ipsilaterally and contralaterally projecting RGCs. Further, a higher proportion of axons projected ipsilaterally in mice lacking CXCL12 or its receptor CXCR4 compared with wild-type mice as a result of misrouting of presumptive contralaterally specified RGC axons. Although RGCs also expressed the alternative CXCL12 receptor ACKR3, the optic chiasm developed normally in mice lacking ACKR3. Our data support a model whereby meningeal-derived CXCL12 helps drive axon growth from CXCR4-expressing RGCs towards the diencephalon midline, enabling contralateral axon growth. These findings further our understanding of the molecular and cellular mechanisms controlling optic pathway development.

BDNF and cAMP are neuroprotective in a porcine model of traumatic optic neuropathy.

Traumatic optic neuropathy (TON) is a devastating condition that can occur after blunt or penetrating trauma to the head, leading to visual impairment or blindness. Despite these debilitating effects, no clinically available therapeutic targets neuroprotection or promotes axon regeneration in this or any optic neuropathy. Limited data in large-animal models are a major obstacle to advancing treatments toward clinical therapeutics. To address this issue, we refined a surgical model of TON in Yucatan minipigs. First, we validated the model by demonstrating visual impairment by flash visual-evoked potential and retinal ganglion cell degeneration and death. Next, we developed and optimized a delivery method and nontoxic dosing of intravitreal brain-derived neurotrophic factor (BDNF) and cAMP. Finally, we showed that intravitreal injection of BDNF and cAMP rescued visual function and protected against retinal ganglion cell death and optic nerve axon degeneration. Together these data in a preclinical large-animal model advance our understanding of and ability to model TON and further identify and develop candidate clinical therapeutics.

Visible-Light Optical Coherence Tomography Fibergraphy of the Tree Shrew Retinal Ganglion Cell Axon Bundles.

We seek to develop techniques for high-resolution imaging of the tree shrew retina for visualizing and parameterizing retinal ganglion cell (RGC) axon bundles in vivo. We applied visible-light optical coherence tomography fibergraphy (vis-OCTF) and temporal speckle averaging (TSA) to visualize individual RGC axon bundles in the tree shrew retina. For the first time, we quantified individual RGC bundle width, height, and cross-sectional area and applied vis-OCT angiography (vis-OCTA) to visualize the retinal microvasculature in tree shrews. Throughout the retina, as the distance from the optic nerve head (ONH) increased from 0.5 mm to 2.5 mm, bundle width increased by 30%, height decreased by 67%, and cross-sectional area decreased by 36%. We also showed that axon bundles become vertically elongated as they converge toward the ONH. Ex vivo confocal microscopy of retinal flat-mounts immunostained with Tuj1 confirmed our in vivo vis-OCTF findings.

Enhanced neuroprotective activity of ophthalmic delivered nerve growth factor conjugated with cell penetrating peptide against optic nerve injury

Nerve growth factor is a well characterized neurotrophic factor that play a critical role in the survival, growth, and differentiation of neurons both in central and peripheral nervous system. However, it is difficult for the conventional exogenous nerve growth factor administration delivery to the central nervous system due to the biological barrier in human bodies. Here, we validated a series of cell penetrating peptides and found that L-PenetraMax significantly enhanced the efficiency of recombinant human nerve growth factor entry into the rat retina. In the optic nerve crush mice model, eye drop administration of recombinant human nerve growth factor alone promoted retinal ganglion cell survival and axon regeneration at high dose, while the combination of recombinant human nerve growth factor with L-PenetraMax significantly enhanced the neuroprotective efficacy at lower dose, thus potentially enhancing the availability of recombinant human nerve growth factor eye drops in patients with optic neuropathy. This study provides the evidence that the noncovalent coadministration of recombinant human nerve growth factor with L-PenetraMax could be a potent strategy for the non-invasive and sustained ocular delivery of therapeutic proteins for improving the optic nerve injury.

Topographic axonal projection at single-cell precision supports local retinotopy in the mouse superior colliculus

Retinotopy, like all long-range projections, can arise from the axons themselves or their targets. The underlying connectivity pattern, however, remains elusive at the fine scale in the mammalian brain. To address this question, we functionally mapped the spatial organization of the input axons and target neurons in the female mouse retinocollicular pathway at single-cell resolution using in vivo two-photon calcium imaging. We found a near-perfect retinotopic tiling of retinal ganglion cell axon terminals, with an average error below 30 μm or 2° of visual angle. The precision of retinotopy was relatively lower for local neurons in the superior colliculus. Subsequent data-driven modeling ascribed it to a low input convergence, on average 5.5 retinal ganglion cell inputs per postsynaptic cell in the superior colliculus. These results indicate that retinotopy arises largely from topographically precise input from presynaptic cells, rather than elaborating local circuitry to reconstruct the topography by postsynaptic cells.