The aberrant retino-retinal projection during optic nerve regeneration in the frog. II. Anterograde labeling with horseradish peroxidase

Previous experiments have shown that a substantial number of regenerating optic axons in adult frogs (Rana pipiens) are misrouted into the opposite optic nerve and retina during early stages of regeneration. This projection is maximal at 5 and 6 weeks after optic nerve crush. To further characterize this anomalous projection, small quantities of horseradish peroxidase (HRP) were injected into the right eye or right optic nerve 5 or 6 weeks after right optic nerve crush. Twenty-four hours later the animals were killed and regenerating axons anterogradely filled with HRP were reacted with the tetramethyl-benzidine method or a diaminobenzidine-CoCl₂ method. Serial reconstruction tracing the course of individual axons through the optic chiasm showed that few of the axons projecting into the opposite optic nerve were collaterals of axons projecting centrally. Instead, the majority of labeled axons misdirected into the opposite nerve or contributing to an expanded projection into the ipsilateral optic tract turned out of the chiasm without branching. Many of the labeled regenerating axons had unusual trajectories within the chiasm, making abrupt turns or changing their direction of growth. Most of the axons misrouted into the opposite nerve came from portions of the chiasm nearest to the nerve of the other eye. In three of eight frogs with an intact optic nerve, a small number of HRP-labeled axons were found in the left nerve after right nerve injection, but there was no indication that these axons reached the left eye. The results from this investigation suggest that the most parsimonious explanation for the chiasmal misrouting of regenerating frog optic axons is that axons are mechanically deflected into inappropriate pathways.     

Denervation of non-optic brain areas along the course of the optic tract does not affect the success of optic nerve regeneration in frogs

Unsuccessful axonal regeneration in frog or goldfish spinal cord is thought to be due in part to inappropriate denervated synaptic sites attracting regenerating axons as they pass by the lesion zone (Bernstein and Bernstein, '69; Bernstein and Gelderd, '73). The reported success of optic nerve regeneration in these same animals may result because there are no inappropriate synaptic sites available to the regenerating axons. To test for this we denervated areas within thalamus and mesencephalon which lie along the path of regenerating optic axons to determine whether or not abnormal connections would form in these areas and affect the success of optic nerve regeneration. After transection of the brainstem through the isthmal region, terminal degeneration was found in several zones adjacent to optic nerve targets or the optic tract (i.e., nucleus rotundus, corpus geniculatum laterale, and torus semicircularis). In adult frogs receiving left isthmal transection, we also crushed the right optic nerve and then examined the regenerated optic projection at intervals up to six months after nerve crush using anterograde transport of ³H-proline. In no instance did the regenerating optic axons alter their distribution within visual neuropil zones or invade those areas deafferented by the isthmal lesion. Histological study showed that the axons cut by the isthmal lesion did not regenerate back to their sites to prevent the invasion of optic axons into these zones. We then attempted to force optic axons into foreign territory by removing a major projection zone, the optic tectum. With tectal ablation and isthmal transection, regenerating optic axons were offered synaptic space made available by both lesions. However, we found abnormal optic projections only in the middle and posterior thalamic neuropil and in the remaining tectal hemisphere. Optic axons did not expand into any of the areas deafferented by the isthmal transection, even though some of them were further denervated by the tectal ablation. We conclude that optic axons will not invade non-optic areas deafferented by an isthmal lesion even if a large number of optic axons have no normal target to innervate.     

Retinal projections throughout optic nerve regeneration in the ornate dragon lizard, Ctenophorus ornatus

In goldfish and frog, optic nerve regeneration is successful, with restoration of retinotopic projections in visual brain centres and the return of functional vision within 1-2 months. By contrast, at 1 year after unilateral optic nerve crush in the ornate dragon lizard (Ctenophorus ornatus), the regenerated retinotectal projections lack topographic order, presumably explaining why the lizards are blind via the experimental eye (Beazley et al. [1997] J. Comp. Neurol. 377:105-120). To determine whether other abnormalities are associated with the inability to restore topographic projections in the lizard, we charted anatomically the time course, accuracy, and stability of optic nerve regeneration by examining visual projections with the lipophillic dye 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI) applied to the optic disk at intervals up to 1 year after optic nerve crush; in addition, DiI tracing of small groups of axons was used to examine the topicity of axons projecting to the tectum. Axons re-innervated visual centres from between 1 and 2 months, a time frame comparable with that in goldfish and frog. However, the projections in lizard were found to differ from those in goldfish and frog in three major ways. First, there was considerable variability within the projection patterns both between individual lizards at any one stage and with time. Second, the projections were inaccurate. As in normal lizards, the major projection was to the contralateral optic tectum, although it lacked detectable retinotopic axon order throughout. Furthermore, misrouting occurred such that regenerating axons formed a persistent projection to the ipsilateral side of the brain that was considerably stronger and more widespread than normal. Minor visual centres also became re-innervated but, in addition, regenerating axons formed persistent projections into the opposite optic nerve and to non-retino-recipient regions such as the nucleus rotundus, hypothalamus, and olfactory nerve, as well as the posterior and tectal commissures. Third, the projections appeared unstable. Projections to both tecta were strongest between 3 and 5 months, but they diminished thereafter. The results suggest that, compared with goldfish and frog, in lizards both pathway and target cues are degraded and/or cannot be read adequately; as a consequence, regenerating axons are unable to navigate exclusively to visual centres and cannot re-form stable connections.     

The changes in neurotrophic properties of the peripheral nerves extracts following blocking of BDNF activity

OBJECTIVES: Retinal ganglion cells (RGCs) of adult rats are unable to regenerate their axons after optic nerve injury and soon after they enter the pathway of apoptosis. They may, however, survive and regenerate new axons in response to application of specific peripheral nerve extracts that presumably contain a range of neurotrophic substances. One of the recognized substances of proven neurotrophic activity is brain-derived neurotrophic factor (BDNF). We have investigated whether blocking the BDNF activity in post-microsomal fractions obtained from 7 day pre-degenerated peripheral nerves would affect its neurotrophic properties towards RGCs after optic nerve transection in adult rats. METHODS: Autologous connective tissue chambers sutured to the distal end of transected optic nerve served as active substances containers. Surviving RGCs were visualized using Dil. The number of myelinated outgrowing fibers within the chambers was evaluated in histologic sections. RESULTS: BDNF and 7 day pre-degenerated nerve extracts, and also extracts with blocked BDNF activity, enhanced RGC fibers outgrowth. The regeneration was significantly weaker in the control group. Blocking the BDNF activity in the 7 day pre-degenerated peripheral nerve extract reduced its neurotrophic effects but the differences were insignificant in comparison with non-blocked extracts. DISCUSSION: The regeneration intensities in groups receiving 7 day pre-degenerated peripheral nerve extracts (PD7) and BDNF were comparable. The number of surviving cells was higher in the PD7 group and there were more regenerating fibers in the BDNF group, which may be explained by the strong BDNF effect on axonal collateralization and sprouting.     

Axonal pathfinding during the regeneration of the goldfish optic pathway

Retinal ganglion cells in fish and amphibians regenerate their axons after transection of the optic nerve. Fiber tracing studies during the third month of regeneration show that the axons have reestablished a basically normal fiber order in the two brachia of the optic tract; axons originating in the ventral hemiretina are concentrated in the dorsal brachium, axons from the dorsal hemiretina in the ventral brachium. Attardi and Sperry (Exp. Neurol. 7:46-64, 1963) first suggested that the reestablishment of the fiber order reflects path-finding by the regenerating axons. Recently, however, Becker and Cook (Development 101:323-337, 1987) have claimed that the fiber order observed at later stages of regeneration is due to secondary axonal rearrangements and that the initial brachial choice is random. In order to evaluate whether regenerating axons are capable of navigating in the optic tract and brachia and on the tectum, the present study examined the pathway choices and the morphology of regenerating axons en route to their tectal targets in goldfish. Subsets of axons were labeled at various time intervals (2 to 30 days) following an optic nerve crush, by intraretinal application of the lipophilic fluorescent tracer 1,1-dioctadecyl-3-3-3'-3'-tetramethylcarbocyanine (DiI). After a survival time of 18 to 72 hours (to allow for diffusion of DiI along the axons), the experimental animals were perfused with fixative and their right and left optic pathways (nerve, tract, and tectum) were dissected free and separated at the chiasm. Fluorescently labeled axons were traced in whole-mounted pathways. Pathway choices were examined at the brachial bifurcation where axons from ventral and dorsal hemiretinae normally segregate. DiI was found to label axons reliably up to their growth cones, even at the earliest stages of regrowth. The pathway choices of the axons were nonrandom. The majority of the ventral axons reached the appropriate, dorsal hemitectum through the appropriate dorsal brachium of the tract. Dorsal axons reached the ventral hemitectum mainly through the ventral brachium. This suggests the presence of specific guidance cues, accessible to the regenerating axons. Differences in the complexity of the growth cones of the regenerating axons (simple in the nerve and tectal fiber layer, complex in the tract and the synaptic layer of the tectum) provide further evidence for specific interactions between the regenerating axons and their substrates along the pathway. These results argue that regenerating retinal axons in fish are capable of axonal path-finding.     

Topographic restriction of TAG-1 expression in the developing retinotectal pathway and target dependent reexpression during axon regeneration

TAG-1, a glycosylphosphatidyl inositol (GPI)-anchored protein of the immunoglobulin (Ig) superfamily, exhibits an unusual spatiotemporal expression pattern in the fish visual pathway. Using in situ hybridization and new antibodies (Abs) against fish TAG-1 we show that TAG-1 mRNA and anti-TAG-1 staining is restricted to nasal retinal ganglion cells (RGCs) in 24- to 72-h-old zebrafish embryos and in the adult, continuously growing goldfish retina. Anti-TAG-1 Abs selectively label nasal RGC axons in the nerve, optic tract, and tectum. Axotomized RGCs reexpress TAG-1, which occurs as late as 12 days after optic nerve lesion, when regenerating RGC axons arrive in the tectum, suggesting TAG-1 reexpression is target contact-dependent. Accordingly, TAG-1 reexpression ceases upon interruption of the regenerating projection by a second lesion. The topographic restriction of TAG-1 expression and its target dependency during regeneration suggests that TAG-1 might play a role in the retinotopic organization and restoration of the retinotectal pathway.     

Thrombospondin expression in nerve regeneration II. comparison of optic nerve crush in the mouse and goldfish

Expression of the extracellular matrix molecule thrombospondin (TSP) was examined following retrobulbar crush injury of the goldfish and mouse optic nerve. TSP was present within the glia limitans and surrounding axon fascicles of the control normal goldfish optic nerve, but was absent from the normal mouse optic nerve. Following crush injury of the goldfish optic nerve, TSP expression increased dramatically along the path of regenerating axons and returned to near normal levels following axonal outgrowth. In contrast, during the unsuccessful attempt at regeneration following crush injury of the mouse optic nerve, TSP expression was present only in glial fibrillary acidic protein (GFAP)-negative, macrophage-rich regions distal to ganglion cell axons. These results indicate that TSP expression is increased in a temporal pattern along the path of regenerating goldfish optic nerve axons and therefore may be involved in successful central nervous system regeneration. The absence of TSP in the environment encountered by damaged mouse optic nerve axons may correlate with the lack of regeneration observed in the mouse optic nerve.     

Lens injury stimulates axon regeneration in the mature rat optic nerve.

In mature mammals, retinal ganglion cells (RGCs) are unable to regenerate their axons after optic nerve injury, and they soon undergo apoptotic cell death. However, a small puncture wound to the lens enhances RGC survival and enables these cells to regenerate their axons into the normally inhibitory environment of the optic nerve. Even when the optic nerve is intact, lens injury stimulates macrophage infiltration into the eye, Müller cell activation, and increased GAP-43 expression in ganglion cells across the entire retina. In contrast, axotomy, either alone or combined with intraocular injections that do not infringe on the lens, causes only a minimal change in GAP-43 expression in RGCs and a minimal activation of the other cell types. Combining nerve injury with lens puncture leads to an eightfold increase in RGC survival and a 100-fold increase in the number of axons regenerating beyond the crush site. Macrophage activation appears to play a key role, because intraocular injections of Zymosan, a yeast cell wall preparation, stimulated monocytes in the absence of lens injury and induced RGCs to regenerate their axons into the distal optic nerve.     

Regeneration of the frog optic nerve

Developing and regenerating frog optic axons grow within optic pathways and form connections only with optic targets. However, unlike normal development, many regenerating optic axons in the adult frog are misrouted within optic pathways, including axons that grow into the opposite retina. Many of the axons misrouted during regeneration appear to be collaterals of axons that grow in normal directions. Ganglion cell loss of up to 60% occurs after optic nerve damage, beginning prior to reinnervation of optic targets. Massive axonal collateralization also takes place near the point of nerve damage, causing the normal order found within the nerve to be lost. Collaterals are eliminated as selective reinnervation is completed, and the smaller complement of optic cell axons remaining after regeneration form an expanded projection within optic targets. Evidence is reviewed that suggests that factors involved in axonal guidance and target recognition during development remain intact in the adult frog brain. Additional conditions resulting from nerve injury causes axonal guidance to be less successful during regeneration.     

Optic nerve regenerates but does not restore topographic projections in the lizard Ctenophorus ornatus

In adult fish and amphibians, the severed optic nerve regenerates and visual behaviour is restored. By contrast, optic axons do not regenerate in the more recently evolved birds and mammals. Here we have investigated optic nerve regeneration in a member of the class Reptilia, phylogenetically intermediate between the fish and amphibians and the birds and mammals. We assessed visual recovery anatomically and behaviourally one year after unilateral optic nerve crush in the adult ornate dragon lizard, Ctenophorus ornatus. Ganglion cell densities and numbers of axons in the optic nerve on either side of the crush site indicated that two-thirds of ganglion cells survived axotomy and regrew their axons. However, myelination fell from a mean of 21% in normals to 5.5% and 3%, proximal and distal to the crush, respectively. Anterograde labelling of the entire optic nerve showed that axons regenerated along essentially normal pathways and that the major projection, as in normals, was to the superficial one-third of the contralateral optic tectum. However, localised retinal injections indicated that regenerated projections lacked retinotopic order. Any one retinal region projected to the entire tectum. This feature presumably explains why the experimental lizards consistently appeared blind to stimuli via the regenerated nerve. Our findings indicate that although axons regenerate along essentially normal pathways in adult lizards, conditions within the visual centres do not allow regenerating optic axons to select appropriate central connections. In a wider context, the result suggests that the ability for regenerating central axons to form topographic maps may also have been lost in the more recently evolved vertebrate classes. J. Comp. Neurol. 377:105-120, 1997. © 1997 Wiley-Liss, Inc.     

Effect of different optic nerve lesions on retinal ganglion cell death in the frog Rana pipiens

Following optic nerve crush in various species of frog, a proportion of the retinal ganglion cells re-establishes functional contact with the optic tectum. However, as much as 50% of the retinal ganglion cells die during this process. The determinants of an individual ganglion cell's fate have not been established. In this study of Rana pipiens, cell survival after optic nerve crush was compared with that after nerve cut followed by stump separation, a procedure that considerably delayed entry of optic axons to the brain. It was also ascertained, in the case of delayed ingrowth, whether application of nerve growth factor immediately after lesion influenced the cell death process. This study confirmed that retinal ganglion cell death is a relatively late event in regeneration, because in several animals where anterograde HRP labeling demonstrated regenerating axons within the tectum, no cell death had occurred. There was no statistically significant difference in cell death at 75 days after lesion between animals receiving nerve crush and those receiving nerve cut with stump separation, even though most crush animals had regenerated a complete visual projection, whereas most nerve cut animals had not. The application of NGF did not influence the level of cell death at 75 days after lesion. These results suggest that contact of optic axons with the optic tract or tectum is not necessary for retinal ganglion cell death to occur. However, this does not necessarily mean that contact with the brain is not involved with cell death during regeneration following nerve crush because it is possible that the mechanisms of cell death are different when axons are prevented from regenerating. Further investigations are therefore required to establish the reasons for this cell death.     

Growth cones of regenerating retinal axons contact a variety of cellular profiles in the transected goldfish optic nerve

Following optic nerve transection in goldfish, retinal axons regenerate. To determine what the growth cones use as a substrate for their growth, regenerating growth cones were labeled by horseradish peroxidase (HRP) application to the retina 5–6 days after intraorbital optic nerve section (ONS) and identified at 10–11 days after ONS in the brain sided (distal) portion of the optic nerve in thick and serial ultrathin sections. Leading growth cones (n = 5) were found in intimate contact with a variety of elements: with myelin fragments alone, with myelin fragments and glial cells, and with the basal lamina of the glia limitans and the surface of a fibroblast outside the boundary of previous fascicles. In ultrathin sections of conventionally treated regenerating optic nerves, (unlabeled) axon profiles—in addition to myelin fragments—were seen to be in contact with an astrocyte and an oligodendrocyte, suggesting that the growth cones of these axons may have been associated with those cells. The data suggest that leading growth cones of regenerating axons may be capable of growing along myelin fragments and on a wide variety of cellular surfaces in the goldfish optic nerve. © 1994 Wiley-Liss, Inc.     

Guiding adult Mammalian sensory axons during regeneration

Misdirection of axons after nerve injury impairs successful regeneration of adult neurons. Investigations of axon guidance in development have provided an understanding of pathfinding, but their relevance to regenerating adult axons is unclear. We investigated adult mammalian axon guidance during regeneration after peripheral nerve injury and focused on the effects of the prototypic guidance molecule nerve growth factor (NGF). Adult rat sensory neurons from dorsal root ganglia that expressed the NGF receptor tropomyosin-related kinase A (trkA) were presented with a point source of NGF in vitro. Naive trkA neurons had no net turning response to NGF, but if they had been preconditioned by a peripheral nerve transection in vivo before culturing, their growth cones were attracted toward the NGF gradient. A laminin substrate was required for this behavior and an anti-trkA antibody interrupted turning. These data demonstrate that injured adult mammalian axons can be guided as they regenerate. Moreover, despite the downregulation of trkA mRNA and protein levels within the dorsal root ganglion after injury, sensory neurons retain and increase trkA protein at the injury site where the regenerating axons are found. This may enhance the axonal response to NGF and allow guidance along an NGF gradient created in vivo in the distal nerve stump.     

Retinal ganglion cell and nonneuronal cell responses to a microcrush lesion of adult rat optic nerve

Injury of the optic nerve has served as an important model for the study of cell death and axon regeneration in the CNS. Analysis of axon sprouting and regeneration after injury by anatomical tracing are aided by lesion models that produce a well-defined injury site. We report here the characterization of a microcrush lesion of the optic nerve made with 10-0 sutures to completely transect RGC axons. Following microcrush lesion, 62% of RGCs remained alive 1 week later, and 28% of RGCs, at 2 weeks. Optic nerve sections stained by hematoxylin-based methods showed a thin line of intensely stained cells that invaded the lesion site at 24 h after microcrush lesion. The lesion site became increasingly disorganized by 2 weeks after injury, and both macrophages and blood vessels invaded the lesion site. The microcrush lesion was immunoreactive for chondroitin sulfate proteoglycans (CSPG), and an adjacent GFAP-negative zone developed early after the lesion, disappearing by 1 week. Luxol fast blue staining showed a myelin-free zone at the lesion site, and myelin remained distal to the lesion at 8 weeks. To study the axonal response to microcrush lesion, anterograde tracing was used. Within 6 h after injury all RGC axons retracted back from the site of lesion. By 1 week after injury, axons regrew toward the lesion, but most stopped abruptly at the injury scar. The few axons that were able to cross the injury site did not extend further in the optic nerve white matter by 8 weeks postlesion. Our observations suggest that both the CSPG-positive scar and the myelin-derived growth inhibitory proteins contribute to the failure of RGC regeneration after injury.     

Distribution of the phosphorylated form of microtubule associated protein 1B in the fish visual system during optic nerve regeneration

Microtubule associated proteins are a heterogeneous group of proteins that have been implicated in regulating microtubule stability. They play an important role in the organisation of the neuronal cytoskeleton during neurite outgrowth, plasticity and regeneration. The fish visual system presents a considerable degree of plasticity. Thus, the retina grows continually throughout life and the optic nerve regenerates after crush. In the present study, we compared the distribution of the microtubule associated protein 1B in its phosphorylated form (MAP1B-phos) in the normal adult fish visual system with that observed during optic nerve regeneration after adult optic nerve crush using a specific monoclonal antibody mAb-150. Expression of MAP1B-phos was observed in some ganglion cell somata and in developing, growing axons within the control optic nerve. Few immunoreactive terminals were seen in the control optic tectum. After optic nerve crush, we found additional MAP1B-phos expression in regenerating axons throughout the visual system. Our results demonstrate that MAP1B-phos is present in growing and regenerating axons of fish retinal ganglion cells, which suggests that the phosphorylated form of MAP1B may play an important role in developmental and regeneration processes within the fish central nervous system.     

Fibroblast growth factor-2 gene delivery stimulates axon growth by adult retinal ganglion cells after acute optic nerve injury

Basic fibroblast growth factor (or FGF-2) has been shown to be a potent stimulator of retinal ganglion cell (RGC) axonal growth during development. Here we investigated if FGF-2 upregulation in adult RGCs promoted axon regrowth in vivo after acute optic nerve injury. Recombinant adeno-associated virus (AAV) was used to deliver the FGF-2 gene to adult RGCs providing a sustained source of this neurotrophic factor. FGF-2 gene transfer led to a 10-fold increase in the number of axons that extended past 0.5 mm from the lesion site compared to control nerves. Detection of AAV-mediated FGF-2 protein in injured RGC axons correlated with growth into the distal optic nerve. The response to FGF-2 upregulation was supported by our finding that FGF receptor-1 (FGFR-1) and heparan sulfate (HS), known to be essential for FGF-2 signaling, were expressed by adult rat RGCs. FGF-2 transgene expression led to only transient protection of injured RGCs. Thus the effect of this neurotrophic factor on axon extension could not be solely attributed to an increase in neuronal survival. Our data indicate that selective upregulation of FGF-2 in adult RGCs stimulates axon regrowth within the optic nerve, an environment that is highly inhibitory for regeneration. These results support the hypothesis that key factors involved in axon outgrowth during neural development may promote regeneration of adult injured neurons.     

Rho kinase is required to prevent retinal axons from entering the contralateral optic nerve

To grow out to contact target neurons an axon uses its distal tip, the growth cone, as a sensor of molecular cues that help the axon make appropriate guidance decisions at a series of choice points along the journey. In the developing visual system, the axons of the output cells of the retina, the retinal ganglion cells (RGCs), cross the brain midline at the optic chiasm. Shortly after, they grow past the brain entry point of the optic nerve arising from the contralateral eye, and extend dorso-caudally through the diencephalon towards their optic tectum target. Using the developing visual system of the experimentally amenable model Xenopus laevis, we find that RGC axons are normally prevented from entering the contralateral optic nerve. This mechanism requires the activity of a Rho-associated kinase, Rock, known to function downstream of a number of receptors that recognize cues that guide axons. Pharmacological inhibition of Rock in an in vivo brain preparation causes mis-entry of many RGC axons into the contralateral optic nerve, and this defect is partially phenocopied by selective disruption of Rock signaling in RGC axons. These data implicate Rock downstream of a molecular mechanism that is critical for RGC axons to be able to ignore a domain, the optic nerve, which they previously found attractive.