Regenerating optic axons restore topography after incomplete optic nerve injury.

Following complete optic nerve injury in a lizard, Ctenophorus ornatus, retinal ganglion cell (RGC) axons regenerate but fail to restore retinotectal topography unless animals are trained on a visual task (Beazley et al. [ 1997] J Comp Neurol 370:105-120, [2003] J Neurotrauma 20:1263-1270). Here we show that incomplete injury, which leaves some RGC axons intact, restores normal topography. Strict RGC axon topography allowed us to preserve RGC axons on one side of the nerve (projecting to medial tectum) while lesioning those on the other side (projecting to lateral tectum). Topography and response properties for both RGC axon populations were assessed electrophysiologically. The majority of intact RGC axons retained appropriate topography in medial tectum and had normal, consistently brisk, reliable responses. Regenerate RGC axons fell into two classes: those that projected topographically to lateral tectum with responses that tended to habituate and those that lacked topography, responded weakly, and habituated rapidly. Axon tracing by localized retinal application of carbocyanine dyes supported the electrophysiological data. RGC soma counts were normal in both intact and axotomized RGC populations, contrasting with the 30% RGC loss after complete injury. Unlike incomplete optic nerve injury in mammals, where RGC axon regeneration fails and secondary cell death removes many intact RGC somata, lizards experience a "win-win" situation: intact RGC axons favorably influence the functional outcome for regenerating ones and RGCs do not succumb to either primary or secondary cell death.

EphA5 and ephrin-A2 expression during optic nerve regeneration: a 'two-edged sword'.

During development, gradients of EphA receptors (nasal(low)-temporal(high)) and their ligands ephrin-As (rostral(low)-caudal(high)) are involved in establishing topography between retinal ganglion cells (RGCs) and the superior colliculus (SC). EphA5-expressing RGC axons are repulsed by ephrin-A2-expressing SC neurones. In adult rats RGCs maintain graded EphA5 expression but ephrin-A2 expression is down-regulated in the SC to a weak gradient. At 1 month after optic nerve transection, EphA5 expression is reduced in the few remaining RGCs and is no longer graded; by contrast, SC ephrin-A2 is up-regulated to a rostral(low)-caudal(high) gradient. Here we examined expression in adult rat 1 month after bridging the retina and SC with a peripheral nerve graft, a procedure that enhances RGC survival and permits RGC axon regeneration. Double labelling with cell markers revealed preservation of a nasal(low)-temporal(high) EphA5 gradient in RGCs and establishment of a rostral(low)-caudal(high) ephrin-A2 gradient within neurones of the SC. The results suggest a potential for guidance cues to restore the topography of RGC axons in the SC. However, high ephrin-A2 levels were also found in astrocytes surrounding the peripheral nerve graft insertion site. The repulsive ephrin-A2 environment offers at least a partial explanation for the observation that only a limited number of RGC axons can exit the graft to enter target central nervous system tissue.

Changing Pax6 expression correlates with axon outgrowth and restoration of topography during optic nerve regeneration.

Pax6, a member of the highly conserved developmental Pax gene family, plays a crucial role in early eye development and continues to be expressed in adult retinal ganglion cells (RGCs). Here we have used Western blots and immunohistochemistry to investigate the expression of Pax6 in the formation and refinement of topographic projections during optic nerve regeneration in zebrafish and lizard. In zebrafish with natural (12-h light/dark cycle) illumination, Pax6 expression in RGCs was decreased during axon outgrowth and increased during the restoration of the retinotectal map. Rearing fish in stroboscopic illumination to prevent retinotopic refinement resulted in a prolonged decrease in Pax6 levels; return to natural light conditions resulted in map refinement and restoration of normal Pax6 levels. In lizard, RGC axons spontaneously regenerate but remain in a persistent state of regrowth and do not restore topography; visual training during regeneration, however, allows a stabilization of connections and return of topography. Pax6 was persistently decreased in untrained animals but remained increased in trained ones. In both species, changes in expression were not due to cell division or cell death. The results suggest that decreased Pax6 expression is permissive for axon regeneration and extensive searching, while higher levels of Pax6 are associated with restoration of topography.

Eph/ephrin expression in the adult rat visual system following localized retinal lesions: localized and transneuronal up-regulation in the retina and superior colliculus.

Following unilateral optic nerve section in adult PVG hooded rat, the axon guidance cue ephrin-A2 is up-regulated in caudal but not rostral superior colliculus (SC) and the EphA5 receptor is down-regulated in axotomised retinal ganglion cells (RGCs). Changes occur bilaterally despite the retino-collicular projection being mostly crossed. Here we investigate the dynamics of Eph/ephrin expression using in situ hybridization and semi-quantitative immunohistochemistry after localized retinal lesions. Unilateral krypton laser lesions to dorso-nasal retina ablated contralaterally projecting RGCs (DN group); ventro-temporal lesions ablated contralaterally and ipsilaterally projecting RGCs (VT group). Lesions of the entire retina served as controls (Total group). Results are compared to normal animals in which tectal ephrin-A2 and retinal EphA5 are expressed, respectively, as shallow ascending rostro-caudal and naso-temporal gradients. In both SCs of DN and Total groups, tectal ephrin-A2 was up-regulated caudally; in the VT group, expression remained normal bilaterally. Unilateral collicular ablation indicated that bilateral changes in ephrin-A2 expression are mediated via intercollicular pathways. EphA5 expression in the VT group was elevated in the intact nasal region of experimental retinae. For each experimental group, EphA5 expression was also elevated in nasal retina of the opposite eye, resulting in uniform expression across the naso-temporal axis. Up-regulation of ephrin-A2 in caudal, but not rostral, SC suggests the enhancement of developmental positional information as a result of injury. Bilateral increases in retinal EphA5 expression demonstrate that signals for up-regulation operate interocularly. The study demonstrates that signals regulating guidance cue expression are both localized and relayed transneuronally.

Generation of transgenic mice with mild and severe retinal neovascularisation.

AIM: To generate a mouse model for slow progressive retinal neovascularisation through vascular endothelial growth factor (VEGF) upregulation. METHODS: Transgenic mice were generated via microinjection of a DNA construct containing the human VEGF165 (hVEGF) gene driven by a truncated mouse rhodopsin promoter. Mouse eyes were characterised clinically and histologically and ocular hVEGF levels assayed by ELISA. RESULTS: One transgenic line expressing low hVEGF levels showed mild clinical changes such as focal fluorescein leakage, microaneurysms, venous tortuosity, capillary non-perfusion and minor neovascularisation, which remained stable up to 3 months postnatal. Histologically, there were some disturbance and thinning of inner and outer nuclear layers, with occasional focal areas of neovascularisation. By contrast, three other lines expressing high hVEGF levels presented with concomitantly severe phenotypes. In addition to the above, clinical features included extensive neovascularisation, haemorrhage, and retinal detachment; histologically, focal to extensive areas of neovascularisation associated with retinal folds, cell loss in the inner and outer nuclear layers, and partial retinal detachment were common. CONCLUSIONS: The authors generated four hVEGF overexpressing transgenic mouse lines with phenotypes ranging from mild to severe neovascularisation. These models are a valuable research tool to study excess VEGF related molecular and cellular changes and provide additional opportunities to test anti-angiogenic therapies.

EphA/ephrin-A interactions during optic nerve regeneration: restoration of topography and regulation of ephrin-A2 expression.

During visual system development, interactions between Eph tyrosine kinase receptors and their ligands, the ephrins, guide retinal ganglion cell (RGC) axons to their topographic targets in the optic tectum. Here we show that Eph/ephrin interactions are also involved in restoring topography during RGC axon regeneration in goldfish. Following optic nerve crush, EphA/ephrin-A interactions were blocked by intracranial injections of recombinant Eph receptor (EphA3-AP) or phospho-inositol phospholipase-C. Topographic errors with multiple inputs to some tectal loci were detected electrophysiologically and increased projections to caudal tectum demonstrated by RT-97 immunohistochemistry. In EphA3-AP-injected fish, ephrin-A2-expressing cells in the retino-recipient tectal layers were reduced in number compared to controls and their distribution was no longer graded. The findings, supported by in vitro studies, implicate EphA/ephrin-A interactions in restoring precise topography and in regulating ephrin-A2 expression during regeneration.

Training on a visual task improves the outcome of optic nerve regeneration.

Optic nerve regeneration in a lizard, Ctenophorus ornatus, is dysfunctional despite survival of most retinal ganglion cells and axon regeneration to the optic tectum. The regenerated retino-tectal projection at 6 months has crude topography but by 1 year is disordered; visually-elicited behavior is absent via the experimental eye. Here, we assess the influence of training on the outcome of optic nerve regeneration. Lizards were trained to catch prey presented within the monocular field of either eye. One optic nerve was then severed and visual stimulation resumed throughout regeneration. In the trained group, presentation was restricted to the eye undergoing optic nerve regeneration; for the untrained group, the unoperated eye was stimulated. Pupil responses returned in trained but not in untrained animals. At 1 year, trained animals oriented to and captured prey; untrained animals demonstrated minimal orienting and failed to capture prey. Regenerated retino-tectal projections were topographic in the trained but not in the untrained group as assessed by in vitro electrophysiological recording and by carbocyanine dye tracing. In vitro electrophysiological recording during application of neurotransmitter antagonists to the tectum revealed that the level of GABAergic inhibition was modest in trained animals but elevated in the untrained group; responses were mainly AMPA-mediated in both groups. We conclude that training improves the behavioral outcome of regeneration, presumably by stabilizing and refining the transient retino-tectal map and preventing a build-up of tectal inhibition. The results suggest that for successful central nerve regeneration to occur in mammals, it may be necessary to introduce training to complement procedures stimulating axon regeneration.

Topographies of retinal cone photoreceptors in two Australian marsupials.

Microspectrophotometry indicates the presence of at least three cone visual pigments in two Australian marsupials, the fat-tailed dunnart (Sminthopsis crassicaudata) and honey possum (Tarsipes rostratus). Here we have examined the distribution of cone types using antisera, JH455 and JH492, that recognize short-wavelength-sensitive (SWS) and medium-to-long-wavelength-sensitive (M/LWS) cone opsins, respectively. SWS cones were concentrated in dorso-temporal retina in the dunnart with a shallow decreasing gradient extending to the periphery (2300-1500/mm2). In the honey possum, SWS cones showed a uniform distribution (2700/mm2), except for a slight increase in a narrow peripheral band (3100/mm2). In both species, M/LWS cones dominated and their distributions were similar to those of retinal ganglion cells: a horizontal streak in the dunnart (31,000-21,000/mm2) and a shallow mid-ventral to peripheral gradient in the honey possum (37,000-26,000/mm2). A low number of cones remained unlabeled when the antisera were combined revealing further minority cone population(s). We discuss cone distributions in relation to visual capabilities and requirements of the species.

PSA-NCAM is up-regulated during optic nerve regeneration in lizard but not in goldfish.

The addition of polysialic acid (PSA) to neural cell adhesion molecule (NCAM) facilitates axon growth. Here we use Western blots and immunohistochemistry to examine expression of PSA-NCAM during optic nerve regeneration. In lizard, retinal ganglion cell axons become transiently PSA-NCAM positive. By contrast, goldfish RGC axons are PSA-NCAM negative both in normal animals and throughout regeneration with the exception of a PSA-NCAM-positive fascicle arising from newly generated RGCs. Transient sialylation of NCAM in lizard may assist regeneration in the nonpermissive reptilian visual pathway and facilitate the reestablishment of a crude topographic map; down-regulation in the long term may contribute to the breakdown in topography. The lack of sialylation in goldfish presumably reflects the permissive nature of the substrate allowing axon regeneration and the successful reestablishment of a topographic map.

Reinnervation of the superior colliculus delays down-regulation of ephrin A2 in neonatal rat.

Although the adult mammalian optic nerve does not regenerate following lesion, in the neonatal rat, retinal ganglion cell (RGC) axons retain the capacity to grow across lesion sites in the brain. Following a brachial lesion at postnatal day 2 (P2), some RGC axons, together with ingrowing cortico-tectal axons, cross the lesion to reinnervate the superior colliculus (SC). Here we use immunohistochemistry to examine expression of the guidance cue ephrin A2 following a brachial lesion. Normal animals show a steady decrease in ephrin A2 immunoreactivity between P5 and P31, with a low rostral to high caudal gradient being evident only at P5. By contrast, after brachial lesion, values are significantly elevated rostrally at P5 and caudally at P12; moreover, a steep rostro-caudal gradient is present at both ages. By P31 values fall to normal levels. Following unilateral enucleation at P2, levels are not significantly different from normal. Our results show that innervation but not denervation triggers increased ephrin A2 expression after a brachial lesion.

Repeated ultrasound guided fetal injections of corticosteroid alter nervous system maturation in the ovine fetus.

INTRODUCTION: Recent studies in sheep have shown that repeated maternal injections of betamethasone are associated with adverse effects within the nervous system. Repeated fetal injections of betamethasone achieve serial improvements in preterm lung function in sheep and are a possible alternative to repeated maternal therapy. We have evaluated the effect of repeated fetal administration of betamethasone on nervous system maturation in an ovine model. METHODS: Date-mated ewes (n = 48) were randomized to receive ultrasound-guided fetal injections of betamethasone or saline between days 104 to 124 of gestation and were delivered by cesarean section on day 125 or 145 (term = 150). Optic and sciatic nerves were prepared for light and electronmicroscopy. Eye diameters were measured and transverse sections of retinae were evaluated. Data were analyzed using a mixed model analysis of variance. RESULTS: Repeated fetal administration of corticosteroid did not significantly affect optic nerve myelination but resulted in significant delays in sciatic axonal growth (p < 0.02) and retinal maturation (p < 0.04). The process of performing repeated fetal injections also significantly affected some retinal parameters. CONCLUSION: Repeated fetal administration of betamethasone alters some aspects of nervous system maturation in sheep. It is premature to plan trials of repeated fetal corticosteroid therapy in humans.

Continued neurogenesis is not a pre-requisite for regeneration of a topographic retino-tectal projection.

Electrophysiological recording demonstrated that visuo-tectal projections are topographically organised after optic nerve regeneration in aged Xenopus laevis. 3H-thymidine autoradiography confirmed previous reports [Taylor, Lack, & Easter, Eur. Journal of Neuroscience 1 (1989) 626-638] that cell division had already ceased at the retinal ciliary margin. The results demonstrate that, contrary to a previous suggestion [Holder & Clarke, Trends in Neuroscience 11 (1988) 94-99], continued neurogenesis is not a pre-requisite for the re-establishment of appropriate connections with target cells.

Pax genes in development and maturation of the vertebrate visual system: implications for optic nerve regeneration.

Pax genes play a pivotal role in development of the vertebrate visual system. Pax6 is the master control gene for eye development: ectopic expression of Pax6 in Xenopus laevis and Drosphila melanogaster leads to the formation of differentiated eyes on the legs or wings. Pax6 is involved in formation of ganglion cells of the retina, as well as cells of the lens, iris and cornea. In addition Pax6 may play a role in axon guidance in the visual system. Pax2 regulates differentiation of the optic disk through which retinal ganglion cell axons exit the eye. Furthermore, Pax2 plays a critical role in development of the optic chiasm and in the guidance of axons along the contralateral or ipsilateral tracts of the optic nerve to visual targets in the brain. During development Pax7 is expressed in neuronal cells of one of the major visual targets in the brain, the optic tectum/superior colliculus. Neurons expressing Pax7 migrate towards the pia and concentrate in the stratum griseum superficiale (SGFS), the target site for retinal axons. Together, expression of Pax2, 6 and 7 may guide axons during formation of functional retinotectal/collicular projections. Highly regulated Pax gene expression is also observed in mature animals. Moreover, evidence suggests that Pax genes are important for regeneration of the visual system. We are currently investigating Pax gene expression in species that display a range of outcomes of optic nerve regeneration. We predict that such information will provide valuable insights for the induction of successful regeneration of the optic nerve and of other regions of the central nervous system in mammals including man.

The development and mature organisation of the end-artery retinal vasculature in a marsupial, the dunnart Sminthopsis crassicaudata.

The end-artery retinal vasculature of a marsupial, the fat-tailed dunnart, was defined by India ink injection and studied in wholemounts. In the adult, the vitreal vasculature supplying the ganglion-cell layer has major paired-vessels in a horizontal H shape. These vessels skirt the area centralis and visual streak that are supplied by fine end-loops. A second vascular layer of uniformly distributed endloops arises from the superficial vessels and lies at the inner nuclear/outer plexiform border. During development, vessels enter the eye via the optic nerve head to form the upper vasculature, assuming an essentially mature arrangement prior to the formation of the area centralis and visual streak. Vessels then descend to form the lower bed. Unlike the cat, the dunnart has retinal vessels that are patent throughout development, their growth is interstitial and reductive remodelling is not seen. A retinal end-artery system may have evolved in marsupials because their precocity requires a vasculature that is functional from early stages of development.

Evidence that regenerating optic axons maintain long-term growth in the lizard Ctenophorus ornatus: growth-associated protein-43 and gefiltin expression.

In the lizard, Ctenophorus ornatus, the optic nerve regenerates but animals remain blind via the experimental eye, presumably as a result of axons failing to consolidate a retinotopic map in the optic tectum. Here we have examined immunohistochemically the expression of the growth-associated protein GAP-43 and the low-molecular-weight intermediate filament protein gefiltin, up to one year after optic nerve crush. Both proteins were found to be permanently up-regulated, suggesting that regenerating axons are held in a permanent state of re-growth. We speculate that, in the lizard, the continued expression of GAP-43 and the failure to switch from the expression of low- to high-molecular-weight intermediate filament proteins are associated with the inability to consolidate a retinotopic projection.

Expression of ephrin-A2 in the superior colliculus and EphA5 in the retina following optic nerve section in adult rat.

The vertebrate retina projects topographically to visual brain centres. In the developing visual system, gradients of ephrins and Eph receptors play a role in defining topography. At maturity, ephrins but not Ephs are downregulated. Here we show that optic nerve section in adult rat differentially regulates the expression of ephrin-A2 in the superior colliculus (SC) and of EphA5 in the retina. Expression was quantified immunohistochemically; ephrin-A2 levels were also estimated by semiquantitative reverse transcriptase polymerase chain reaction. In the normal SC, ephrin-A2 was expressed at low levels. At 1 month, levels of protein and of mRNA were upregulated across the contralateral SC giving rise to an increasing rostro-caudal gradient. At 6 months, levels had fallen but a gradient remained. In the retina of normal animals, EphA5 was expressed as an increasing naso-temporal gradient. By 1 month, expression was decreased in far temporal retina, resulting in a uniform expression across the naso-temporal axis. We suggest that denervation-induced plastic changes within the SC modify expression of these molecules.

Topographic order of retinofugal axons in a marsupial: implications for map formation in visual nuclei.

We studied axon order in the primary visual pathway and in nine retinorecipient nuclei of a small marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata) using animals at postnatal day (P) 40 and P80. Dorsal, ventral, nasal, and temporal axons enter the optic nerve true to their retinal origin being respectively dorsal, ventral, medial, and lateral; the arrangement is retained to the chiasm. Dorsal and ventral axons maintain their respective locations within the chiasm but at the base of the contralateral optic tract undergo a 180 degrees axial rotation, thus reversing the dorsoventral axis with respect to the retina. The alignment is conserved along the optic tract with dorsal and ventral axons mapping directly into appropriate quadrants of each retinorecipient nucleus. Nasal and temporal axons remain segregated as they decussate and lie respectively superficially and deep along the optic tract but with some intermingling. Within each retinorecipient nucleus, the nasotemporal axis is clearly demarcated, being represented in either a rostrocaudal (ventral and dorsal lateral geniculate nuclei; lateral posterior, dorsal terminal, and pretectal nuclei) or caudorostral (medial terminal and caudal pretectal nuclei, intergeniculate nucleus and superior colliculus) direction. The results imply that the dorsoventral axis in the retinorecipient nuclei could be due to preordering within the pathway, whereas the nasotemporal axis is determined by target-based cues. Moreover, cues for the orientation of the nasotemporal axis within retinorecipient nuclei must be localised within individual nuclei rather than as a single organiser, as previously envisaged (Chung and Cooke [1978] Proc. R. Soc. Lond. B. 210:335-373).

Transient up-regulation of the rostrocaudal gradient of ephrin A2 in the tectum coincides with reestablishment of orderly projections during optic nerve regeneration in goldfish.

During development, a graded expression of ephrin A2 has been implicated in retinotectal map formation. Here we have examined ephrin A2 expression during optic nerve regeneration in the mature goldfish. In the tecta of normal animals, a gradient of ephrin A2 expression is detected in cell bodies within the stratum fibrosum et griseum superficiale with more immunopositive cells caudally than rostrally. The gradient in the mature animal presumably reflects the plasticity associated with continued retinal and tectal neurogenesis. During optic nerve regeneration, expression throughout the tectum is increased by 1 month as a strong rostrocaudal gradient. The gradient declines to normal by 3 months. The up-regulation of ephrin A2 during optic nerve regeneration is likely to be instrumental in reestablishing the retinotectal map.

Visual system in a diurnal marsupial, the numbat (Myrmecobius fasciatus): retinal organization, visual acuity and visual fields.

The visual system of the numbat (Myrmecobius fasciatus), a highly endangered diurnal marsupial, has been investigated both anatomically and behaviorally. The ganglion cell layer, examined in cresyl-violet stained wholemounts, contained 832,800 ganglion cells; the number of ganglion cells corresponded to optic axon counts. An area centralis was located in the mid-temporal retina, where cells formed a bilayer, but there was no evidence of a visual streak. Visual acuity, estimated from counts of peak ganglion cell density (8,100/mm(2)) and measurements of posterior nodal distance (7.84 mm), was found to be 6.3 cycles per degree. The value was similar to that of 5.2 cycles per degree estimated by behavioral tests. Sectioned material revealed the presence of numerous oil-droplets in a cone-dominated retina. A rich retinal vasculature of the end-artery type of paired arteries and veins formed beds in the ganglion cell and inner nuclear layers. Measurements of visual fields revealed a binocular overlap of 80 degrees in the horizontal plane, and a total field of view approaching 240 degrees of visual angle. Monitoring of pupillary responses with an infrared video camera showed that the numbat possesses a remarkably wide, static pupil. Our results are discussed in relation to the ecology of the species and its phylogeny.

Retinal maturation is delayed by repeated, but not single, maternal injections of betamethasone in sheep.

PURPOSE: The safety and efficacy of prescribing a single maternal course of corticosteroid during pregnancy has been documented in human trials. However, the current trend is to prescribe repeated courses of corticosteroid. We investigated an aspect of the safety of this practice in an animal model. METHODS: Date-mated ewes received saline, single or four corticosteroid injections between days 104 and 124 of gestation (term = 150). Lambs were delivered on day 125 or 145 by caesarian section after spinal anaesthesia. Eye diameters were measured and semi-thin toluidine-blue-stained transverse sections of retinae were analysed using an Optimus Image Analysis program. RESULTS: At 125 days, retinal measures in the ventral periphery and area centralis were significantly thinner than control (p = 0.0001). At 145 days, total eye size was significantly reduced compared with control (p = 0.03), and retinal measures in the ventral periphery (p = 0.0001), but not the area centralis (p = 0.19), remained significantly different from control. CONCLUSION: Repeated maternal administration of corticosteroid may affect retinal maturation in the fetus.