Growth preferences of adult rat retinal ganglion cell axons in retinotectal cocultures

We examined whether regenerating axons from adult rat ganglion cells are able to recognize their appropriate target region in vitro. Explants from adult rat retina were cocultured with embryonic sagittal midbrain slices in Matrigel®. The midbrain sections contained the superior colliculus, the main target for retinal ganglion cell axons in rats, and the inferior colliculus. We observed a statistically significant preference of both temporal and nasal retinal axons to grow toward their appropriate target region (anterior and posterior superior colliculus, respectively). No preferential growth of retinal ganglion cell axons was detected in controls, for which retinal explants were cultured on their own. When retinal ganglion cell axons were given a choice between superior colliculus and inferior colliculus, axons from nasal retina preferentially grew toward the posterior superior colliculus and avoided the inferior colliculus. In contrast, temporal axons in the same assay did not show preference for either of the colliculi. These findings suggest that regenerating axons from adult rat retina are able to recognize target-specific guidance cues released from embryonic midbrain targets in vitro. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 379–387, 1998     

Formation of functional synapses by regenerating adult rat retinal ganglion cell axons in midbrain target regions in vitro

The ability of adult rat retinal ganglion cell (RGC) axons to reinnervate normal target regions was examined in vitro. In co-culture experiments, adult rat retinal explants were placed adjacent to fetal rat midbrain sections that contained the superior colliculus (SC) which is the main target for RGC axons. Adult rat RGCs regrew axons over more than 500 μm on a polylysine-laminin substrate to reach the co-cultured explants. By using neurofilament immunohistochemistry and the fluorescent dye Dil for anterograde and retrograde tracing, it was shown that (1) adult rat RGCs with a stereotyped morphology survived in explant cultures for more than 4 weeks in the presence of fetal midbrain explants, (2) regenerating RGC axons preferentially terminated within midbrain target regions, and (3) RGCs formed functional synapses. In addition, the maturation of the SC region in midbrain explants was examined histologically and ultrastructurally to demonstrate appropiate target development. © 1993 John Wiley & Sons, Inc.     

Cyclic AMP and the regeneration of retinal ganglion cell axons

In this paper we present a brief review of studies that have reported therapeutic benefits of elevated cAMP on plasticity and regeneration after injury to the central nervous system (CNS). We also provide new data on the cellular mechanisms by which elevation of cyclic adenosine monophosphate (cAMP) promotes cytokine driven regeneration of adult CNS axons, using the visual system as the experimental model. cAMP is a second messenger for many intracellular signalling pathways. Elevation of cAMP in the eye by intravitreal injection of the cell permeant analogue (8-(4-chlorophenylthio)-adenosine-3′,5′-cyclic monophosphate; CPT-cAMP), when added to recombinant ciliary neurotrophic factor (rCNTF), significantly enhances rCNTF-induced regeneration of adult rat retinal ganglion cell (RGC) axons into peripheral nerve (PN) grafted onto transected optic nerve. This effect is mediated to some extent by protein kinase A (PKA) signalling, but CPT-cAMP also acts via PI3K/Akt signalling to reduce suppressor of cytokine signalling protein 3 (SOCS3) activity in RGCs. Another target for cAMP is the exchange protein activated by cAMP (Epac), which can also mediate cAMP-induced axonal growth. Here we describe some novel results and discuss to what extent the pro-regenerative effects of CPT-cAMP on adult RGCs are mediated via Epac as well as via PKA-dependent pathways. We used the established PN–optic nerve graft model and quantified the survival and regenerative growth of adult rat RGCs after intravitreal injection of rCNTF in combination with a selective activator of PKA and/or a specific activator of Epac. Viable RGCs were identified by βIII-tubulin immunohistochemistry and regenerating RGCs retrogradely labelled and quantified after an injection of fluorogold into the distal end of the PN grafts, 4 weeks post-transplantation. The specific agonists of either PKA or Epac were both effective in enhancing the effects of rCNTF on RGC axonal regeneration, but interestingly, injections that combined rCNTF with both agonists were significantly less effective. The results are discussed in relation to previous CPT-cAMP studies on RGCs, and we also consider the need to modulate cAMP levels in order to obtain the most functionally effective regenerative response after CNS trauma.This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation.     

Embryonic lens repels retinal ganglion cell axons.

During development of the vertebrate visual system, retinal ganglion cell (RGC) axons follow a precise path toward their midbrain targets. Although much is known about the cues that direct RGC axons once they have left the optic disc, less is known about the guidance of axons at earlier stages, when RGCs first send out their axons to navigate within the developing retina. Using collagen gel coculture experiments, we find that the embryonic lens produces a powerful diffusible repulsive activity for RGC axons. We also find that this activity is localized to the lens epithelium and not the lens fiber layer, while the pigmented epithelium and vitreous humour are devoid of activity. The further observation that the lens also chemorepels primary sensory axons, but does not repel olfactory bulb axons, shows that this activity is specific for subsets of axons. Our experiments have excluded two candidate repellents for RGC axons (collapsin-1/sema III and chondroitin sulfate proteoglycans). These results implicate the lens in the earliest stages of RGC axon guidance. One function of the lens repellent may be to prevent aberrant targeting toward the lens, and it may also be involved in the directional guidance of RGC axons toward the optic disc.     

Complement-mediated microglial clearance of developing retinal ganglion cell axons

In many parts of the developing vertebrate nervous system, axons are pruned to establish mature patterns of connectivity. In this issue of Neuron, Schafer et al. (2012) show that microglia may play a role in developmental axon pruning in the thalamus by engulfing presynaptic retinal ganglion cell terminals via a C3- and CR3-dependent mechanism.     

Weak electric fields serve as guidance cues that direct retinal ganglion cell axons in vitro

Growing axons are directed by an extracellular electric field in a process known as galvanotropism. The electric field is a predominant guidance cue directing retinal ganglion cell (RGC) axons to the future optic disc during embryonic development. Specifically, the axons of newborn RGCs grow along the extracellular voltage gradient that exists endogenously in the embryonic retina (Yamashita, 2013 [8]). To investigate the molecular mechanisms underlying galvanotropic behaviour, the quantification of the electric effect on axon orientation must be examined. In the present study, a culture system was built to apply a constant, uniform direct current (DC) electric field by supplying an electrical current to the culture medium, and this system also continuously recorded the voltage difference between the two points in the medium. A negative feedback circuit was designed to regulate the supplied current to maintain the voltage difference at the desired value. A chick embryo retinal strip was placed between the two points and cultured for 24 h in an electric field in the opposite direction to the endogenous field, and growing axons were fluorescently labelled for live cell imaging (calcein-AM). The strength of the exogenous field varied from 0.0005 mV/mm to 10.0 mV/mm. The results showed that RGC axons grew in the reverse direction towards the cathode at voltage gradients of ≥0.0005 mV/mm, and straightforward extensions were found in fields of ≥0.2–0.5 mV/mm, which were far weaker than the endogenous voltage gradient (15 mV/mm). These findings suggest that the endogenous electric field is sufficient to guide RGC axons in vivo.     

The outgrowth response of the axons of developing and regenerating rat retinal ganglion cells in vitro to neurotrophin treatment

BDNF and NT-4 (but not NT-3 or CNTF) significantly enhanced the outgrowth of early embryonic and adult regenerating RGC axons when provided with a supportive substrate in vitro. BDNF and NT-4 treatment transiently increased RGC axon outgrowth from E15 rat retinas but not from retinas at older embryonic ages. The transient effect of BDNF and NT-4 and the inability of the neurotrophins to promote outgrowth from older embryonic retinal explants suggests a time frame of neurotrophin action and that other chemical factors (target-derived or otherwise) may be necessary for the continued maintenance of developing RGC axons. BDNF and NT-4 also enhanced the outgrowth of regenerating axons from adult retinal explants, but appeared to have a more subtle effect on axon outgrowth, in that the growth-promoting effects of BDNF and NT-4 appeared continuous throughout the incubation period. The suppression of RGC axon outgrowth from embryonic and adult retinae cultured in trkB-IgG-containing medium suggests that the response of developing and regenerating axons, to BDNF and NT-4 are likely to occur through trkB signalling.     

Topographic targeting and pathfinding errors of retinal axons following overexpression of ephrinA ligands on retinal ganglion cell axons

In the retinotectal projection, the Eph receptor tyrosine kinase ligands ephrinA2 and ephrinA5 are differentially expressed not only in the tectum, but also in a high-nasal-to-low-temporal pattern in the retina. Recently, we have shown that retrovirally driven overexpression of ephrinA2 on retinal axons leads to topographic targeting errors of temporal axons in that they overshoot their normal termination zones in the rostral tectum and project onto the mid- and caudal tectum. The behavior of nasal axons, however, was only marginally affected. Here, we show that overexpression of ephrinA5 affects the topographic targeting behavior of both temporal and nasal axons. These data reinforce the idea that differential ligand expression on retinal axons contributes to topographic targeting in the retinotectal projection. Additionally, we found that ectopic expression of ephrinA2 and ephrinA5 frequently leads to pathfinding errors at the chiasm, resulting in an increased stable ipsilateral projection.     

The directed growth of retinal axons towards surgically transposed tecta in Xenopus; an examination of homing behaviour by retinal ganglion cell axons

The growth of optic axons towards experimentally rotated tecta has been studied. In stage 24/25 embryos, a piece of the dorsal neural tube, containing the dorsal midbrain rudiment, was rotated through 180 degrees. At later stages of development, the pathways of growing optic axons were investigated by labelling with either horseradish peroxidase or fluorescent dye. It is shown that retinal ganglion cell axons followed well-defined pathways, in spite of the abnormal structure of the brain, and were able to locate displaced tecta. This directed outgrowth of retinal axons in the optic tracts appears to be related either to the tectum or to some other component included in the graft operations. In tadpoles in which the midbrain rudiment was removed, optic axons still followed the normal course of the optic tract. This observation argues against long-range target attraction as being essential in guiding growing retinal axons towards the tectum. An alternative axon guidance mechanism, selective fasciculation, is discussed as a possible alternative to explain the directed axon outgrowth which occurs in both the normal and in these experimentally manipulated tadpoles.     

ATP and calmodulin dependent actomyosin aggregates induced by cytochalasin D in goldfish retinal ganglion cell axons in vitro

Growing retinal ganglion cell (RGC) axons of the goldfish have mobile varicosities, which play a role in rapid bulk redistribution of axoplasm (Koenig, Kinsman, Repasky, and Sultz, 1985; Edmonds and Koenig, 1987). Varicosities contain a tubulo-vesicular SER embedded in an actin-containing cytomatrix (Koenig et al., 1985). Cytochalasin D (CD) induces the formation of focal cytoskeletal aggregates throughout preterminal axons and especially in varicosities. The aggregates are visible when labelled with fluoroscein isothiocyanate (FITC)-conjugated phalloidin. Double-labelling experiments show that Texas red-myosin or rhodamine isothiocyanate (RITC)-calmodulin immunofluorescence co-localizes with FITC-phalloidin-labelled aggregates. Formation of aggregates is blocked by calmidazolium, a calmodulin antagonist. Axon models permeabilized with digitonin retain the capacity to form focal aggregates in response to CD, when ATP or adenosine-5'-O(3-thiotriphosphate) (ATP-gamma S) is present in the permeabilization buffer, but not when 5'-adenylylimidodiphosphate (AMP-PNP) is present. The latter result indicates that formation of focal aggregates depends on ATP. The findings suggest that the formation of focal aggregates in immature axons is a manifestation of actomyosin interactions after free actin-filament ends are generated by CD treatment.     

Role of cell adhesion molecule DM-GRASP in growth and orientation of retinal ganglion cell axons

The cell adhesion molecule (CAM) DM-GRASP was investigated with respect to a role for axonal growth and navigation in the developing visual system. Expression analysis reveals that DM-GRASP's presence is highly spatiotemporally regulated in the chick embryo retina. It is restricted to the optic fiber layer (OFL) and shows an expression maximum in a phase when the highest number of retinal ganglion cell (RGC) axons extend. In the developing retina, axons grow between the DM-GRASP-displaying OFL and the Laminin-rich basal lamina. We show that DM-GRASP enhances RGC axon extension and growth cone size on Laminin substrate in vitro. Preference assays reveal that DM-GRASP-containing lanes guide RGC axons, partially depending on NgCAM in the axonal membrane. Inhibition of DM-GRASP in organ-cultured eyes perturbs orientation of RGC axons at the optic fissure. Instead of leaving the retina, RGC axons cross the optic fissure and grow onto the opposite side of the retina. RGC axon extension per se and navigation from the peripheral retina towards the optic fissure, however, is not affected. Our results demonstrate a role of DM-GRASP for axonal pathfinding in an early phase of the formation of the higher vertebrate central nervous system.     

Spermidine promotes retinal ganglion cell survival and optic nerve regeneration in adult mice following optic nerve injury

Spermidine acts as an endogenous free radical scavenger and inhibits the action of reactive oxygen species. In this study, we examined the effects of spermidine on retinal ganglion cell (RGC) death in a mouse model of optic nerve injury (ONI). Daily ingestion of spermidine reduced RGC death following ONI and sequential in vivo retinal imaging revealed that spermidine effectively prevented retinal degeneration. Apoptosis signal-regulating kinase-1 (ASK1) is an evolutionarily conserved mitogen-activated protein kinase kinase kinase and has an important role in ONI-induced RGC apoptosis. We demonstrated that spermidine suppresses ONI-induced activation of the ASK1-p38 mitogen-activated protein kinase pathway. Moreover, production of chemokines important for microglia recruitment was decreased with spermidine treatment and, consequently, accumulation of retinal microglia is reduced. In addition, the ONI-induced expression of inducible nitric oxide synthase in the retina was inhibited with spermidine treatment, particularly in microglia. Furthermore, daily spermidine intake enhanced optic nerve regeneration in vivo. Our findings indicate that spermidine stimulates neuroprotection as well as neuroregeneration, and may be useful for treatment of various neurodegenerative diseases including glaucoma.Traumatic optic neuropathy is a common clinical problem that occurs in 0.5–5% of patients with closed head injury.¹ A damage to the optic nerve causes shear stress and induces secondary swelling within the optic canal, accompanied by subsequent RGC loss and optic nerve atrophy.² Although no large natural history or randomized controlled trial has been published, neither corticosteroid therapy nor optic canal decompression surgery is considered as standard treatments for patients with traumatic optic neuropathy,³ and there is a lack of effective treatment at present. Research into finding therapeutic targets for treatment of traumatic optic neuropathy indicated that neuroprotection and axon regeneration may be effective strategies and studies using an optic nerve injury (ONI) model in rodents have provided useful information. For example, neurotrophins, such as brain-derived neurotrophic factor and ciliary neurotrophic factor, protect retinal ganglion cells (RGCs) and promote axon regeneration in an ONI model.^{4, 5, 6} In addition, inhibition of neuroinflammatory events such as upregulation of tumor necrosis factor (TNF)-α and nitric oxide synthase (NOS) may be effective for RGC protection following ONI.⁷ The ONI model mimics some aspects of glaucoma, including RGC death induced by excitotoxicity and oxidative stress, and therefore it is also a useful animal model for glaucoma.Glaucoma is one of the leading causes of vision loss in the world and it is estimated that this condition will affect more than 80 million individuals worldwide by 2020, with at least 6–8 million individuals becoming bilaterally blind.⁸ Glaucoma is characterized by progressive degeneration of RGCs and their axons, which are usually associated with elevated intraocular pressure, but there is a subset of glaucoma termed normal tension glaucoma (NTG) that presents with statistically normal intraocular pressure. There are several animal models of glaucoma, including DBA/2J mice,⁹ and inducible models such as cauterization of episcleral veins.^{10, 11, 12} In addition, we previously reported that loss of glutamate transporters (EAAC1 or GLAST) in mice leads to RGC degeneration that is similar to NTG¹³ and these animal models have been useful in examining potential therapeutic targets.^{14, 15, 16}Spermidine is naturally and almost exclusively accumulated in glial cells in the brain and retina.^{17, 18} It acts as an endogenous free radical scavenger and inhibits the action of reactive oxygen species. Indeed, it has been reported that spermidine has key roles in mediating protection against oxidative damage caused by hydrogen peroxide in cultured mouse fibroblasts¹⁹ and administration of spermidine extended the lifespan of yeast, flies, worms and human immune cells by upregulating the lysosomal/vacuolar degradation pathway, referred to as autophagy, which leads to enhanced resistance to oxidative stress and decreased cell death.²⁰ Previously, we reported that oral administration of spermidine ameliorates severity of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, by suppression of oxidative stress,²¹ suggesting that spermidine may also be effective in protecting RGCs from increased oxidative stress associated with various pathogenic conditions in the eye including traumatic optic neuropathy and glaucoma.In this study, we examined the effects of daily spermidine intake on ONI-induced retinal degeneration. We monitored changes in retinal morphology over a course of 2 weeks following ONI, using optical coherence tomography (OCT), which permits noninvasive, longitudinal and quantitative assessment of retinal structures in living animals. We also explored possible mechanisms associated with spermidine-mediated neuroprotection.     

Gap-43 immunoreactivity and axon regeneration in retinal ganglion cells of the rat

Retinal ganglion cells (RGCs) in rats were retrogradely labeled with the fluorescent tracer Fluorogold (FG) and subjected to GAP-43 and c-JUN immunocytochemistry to identify those RGSs that are capable of regenerating an axon. After optic nerve section (ONS) and simultaneous application of FG to the nerve stump (group 1 experiments), GAP-43 immunoreactive RGCs (between 2 and 21 days after ONS) always represented a subfraction of both FG-labeled (i.e., surviving) RGCs and RGCs exhibiting c-JUN. GAP-43 immunoreactive RGCs represented 22% of RGCs normally present in rat retinae and 25% of surviving RGCs at 5 days after ONS but were reduced to 2% and 1%, which is 6% and 5% of survivors at 14 and 21 days, respectively. In animals that received a peripheral nerve (PN) graft after ONS (group 2 experiments), RGCs with regenerating axons were identified by FG application to the graft at 14 and 21 days. When examined at 21 and 28 days, all FG-labeled RGCs exhibited GAP-43 immunoreactivity, and FG/GAP-43-labeled RGCs were 3% and 2% of those resent in normal rat retinae. In relation to surviving. RGCs GAP-43 immunoreactive RGCs represented 10% at both time points. FG-/GAP-43 labeled RGCs also exhibited c-JUN, but c-JUN immunoreactive RGCs were at both time points at least twice as numerous a FG-/GAP-43-labeled RGCs. These data suggest that regenerating axons in PN grafts derive specifically from GAP-43 reexpressing RGCs. Appearance of GAP-43 immunoreactivity may therefore identify those RGCs that are capable of axonal regeneration or sprouting. 1994 John Wiley & Sons, Inc.     

The cell adhesion molecule NrCAM is crucial for growth cone behaviour and pathfinding of retinal ganglion cell axons

We investigated the role of the cell adhesion molecule NrCAM for axonal growth and pathfinding in the developing retina. Analysis of the distribution pattern of NrCAM in chick embryo retina sections and flat-mounts shows its presence during extension of retinal ganglion cell (RGC) axons; NrCAM is selectively present on RGC axons and is absent from the soma. Single cell cultures show an enrichment of NrCAM in the distal axon and growth cone. When offered as a substrate in addition to Laminin, NrCAM promotes RGC axon extension and the formation of growth cone protrusions. In substrate stripe assays, mimicking the NrCAM-displaying optic fibre layer and the Laminin-rich basal lamina, RGC axons preferentially grow on NrCAM lanes. The three-dimensional analysis of RGC growth cones in retina flat-mounts reveals that they are enlarged and form more protrusions extending away from the correct pathway under conditions of NrCAM-inhibition. Time-lapse analyses show that these growth cones pause longer to explore their environment, proceed for shorter time spans, and retract more often than under control conditions; in addition, they often deviate from the correct pathway towards the optic fissure. Inhibition of NrCAM in organ-cultured intact eyes causes RGC axons to misroute at the optic fissure; instead of diving into the optic nerve head, these axons cross onto the opposite side of the retina. Our results demonstrate a crucial role for NrCAM in the navigation of RGC axons in the developing retina towards the optic fissure, and also for pathfinding into the optic nerve.     

Immortalized olfactory ensheathing glia promote axonal regeneration of rat retinal ganglion neurons

Olfactory bulb ensheathing glia (OEG) have attracted special attention during the last few years because of their unique properties in promoting regeneration of adult mammalian central nervous system (CNS) components. However the molecular and cellular characteristics responsible for this capacity remain to be revealed. Such studies are presently hindered by the lack of a plentiful source of homogenous OEG. Thus the availability of immortalized OEG lines maintaining the regenerative characteristics of the primary cultures would represent an unlimited source of OEG for use not only in biochemical analyses of neuroregenerative mechanisms but also to characterize their regenerative properties in models in culture and in vivo. We have immortalized primary rat OEG using the SV40 large T antigen expressed from a constitutive cellular promotor, and report here the isolation and characterization of clonal lines. These OEG clonal lines were comparable to primary OEG and Schwann cells in the promotion of axonal regeneration of mature rat retinal ganglion neurons (RGN) but, significantly, this culture assay system more closely reflects the in vivo reparative properties of OEG on transected nerves than other assays of neuritogenesis in that it revealed OEG cells to promote the growth of a larger number of long axons than Schwann cells. Using this assay we were able to grade our OEG lines for their neuroregenerative capacity, opening the possibility of identifying molecules with correlative expression levels in these cells. Our preliminary characterization revealed that the expression level of a classical OEG marker, the p75-NGF receptor, does not correlate with neuroregenerative capacity.     

Neurotrophin-4/5 (NT-4/5) increases adult rat retinal ganglion cell survival and neurite outgrowth in vitro

Retinal ganglion cell (RGC) survival and neurite outgrowth were investigated in retinal explants from adult rats. Neutrotrophin-4/5 (NT-4/5) caused dose-dependent increases in neurite outgrowth with one-half maximal effects at approximately 0.5 ng/ml and maximal effects at 5 ng/ml. In explants treated for 7 days, the actions of NT-4/5 were similar to those of brain-derived neurotrophic factor (BDNF); with either neurotrophin, nearly twice as many RGCs survived and there was a two- to threefold increase in the number of neurites formed by RGCs. Combinations of saturating concentrations of NT-4/5 and BDNF did not enhance these in vitro effects, implying that both neurotrophins share a common signaling pathway. In contrast, nerve growth factor (NGF), neurotrophin-3 (NT-3), or ciliary nuerotrophic factor (CNTF) appeared to exert minimal influences on RGC survival or neurite outgrowth. 1994 John Wiley & Sons, Inc.     

Postsynaptic neuronal activity promotes regeneration of retinal axons

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The polysialic acid moiety of the neural cell adhesion molecule is involved in intraretinal guidance of retinal ganglion cell axons

We have characterized the antigen recognized by mab10, a monoclonal antibody that has been shown to modify outgrowth of thalamic and cortical axons in vitro, and investigated the influence of this antibody on axonal growth in the chicken retina in vivo. Immunopurification, peptide sequencing, and biochemical characterization proved the epitope recognized by mab10 to be polysialic acid (PSA), associated with the neural cell adhesion molecule (NCAM). Intravitreal injections of antibody-secreting hybridoma cells were combined with whole-mount studies using the fluorescent tracer 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI). Pathfinding at the optic fissure was affected, resulting in a failure of axons to exit into the nerve. Misprojections also occurred in more peripheral areas of the retina; however, axons eventually oriented toward the center. Similar projection errors were observed after enzymatic removal of PSA by injecting endoneuraminidase N (endo N). Quantitative measurements of the optic nerve diameter as well as the width of the optic fiber layer confirmed that many axons failed to leave the retina and grew back in the optic fiber layer of the retina. Our findings suggest that NCAM-linked PSA is involved in guiding ganglion cell axons in the retina and at the optic fissure.