Regenerating Fish Optic Nerves Research Articles

Schwann cells in the regenerating fish optic nerve: evidence that CNS axons, not the glia, determine when myelin formation begins.

Fish optic nerve fibres quickly regenerate after injury, but the onset of remyelination is delayed until they reach the brain. This recapitulates the timetable of CNS myelinogenesis during development in vertebrate animals generally, and we have used the regenerating fish optic nerve to obtain evidence that it is the axons, not the myelinating glial cells, that determine when myelin formation begins. In fish, the site of an optic nerve injury becomes remyelinated by ectopic Schwann cells of unknown origin. We allowed these cells to become established and then used them as reporters to indicate the time course of pro-myelin signalling during a further round of axonal outgrowth following a second upstream lesion. Unlike in the mammalian PNS, the ectopic Schwann cells failed to respond to axotomy and to the initial outgrowth of new optic axons. They only began to divide after the axons had reached the brain. Shortly afterwards, small numbers of Schwann cells began to leave the dividing pool and form myelin sheaths. More followed gradually, so that by 3 months remyelination was almost completed and few dividing cells were left. Moreover, remyelination occurred synchronously throughout the optic nerve, with the same time course in the pre-existing Schwann cells, the new ones that colonised the second injury, and the CNS oligodendrocytes elsewhere. The optic axons are the only common structures that could synchronise myelin formation in these disparate glial populations. The responses of the ectopic Schwann cells suggest that they are controlled by the regenerating optic axons in two consecutive steps. First, they begin to proliferate when the growing axons reach the brain. Second, they leave the cell cycle to differentiate individually at widely different times during the ensuing 2 months, during the critical period when the initial rough pattern of axon terminals in the optic tectum becomes refined into an accurate map. We suggest that each axon signals individually for myelin ensheathment once it completes this process.

New light on nerve regeneration in the mammalian nervous system

It has long been known that while damaged nerves of the peripheral nervous system in mammals are able to regenerate, this does not occur in the central nervous system. Recent studies have shown that a chemical factor present in regenerating fish optic nerves facilitates regeneration of nerve axons in rodents. Further research encourages the hope that this may ultimately lead to the successful treatment of nerve damage in humans.

L1 immunoreactivity in the developing fish visual system

Previous studies have suggested that L1, the cell adhesion molecule, is present in the regenerating fish optic nerve. The present study was undertaken in order to determine whether L1 is expressed by fish neurons and specifically by non-myelinated axons, using fish retinal explants in vitro and the developing fish visual system in vivo. In vitro, the nonmyelinated axons emerging from retinal explants showed L1 immunoreactivity and in vivo, L1 immunoreactive sites were found to be associated with areas rich in non-myelinated axons. At embryonic stage 23, as the eye developed and optic nerve axons began to elongate towards the tectum, L1-like immunoreactivity was seen both in optic nerve and in plexiform layers of the retina. At this stage and until hatching, the cellular layers within the retina showed little or no staining relative to the layers containing axons or dendrites. After hatching, L1 immunoreactivity was also observed in the ganglion cell layer, but upon maturation both the retina and the optic nerve lost most of their L1 immunoreactivity. We therefore suggest that non-myelinated axons of the fish visual system express L1 during development, which is lost after myelination and presumably reappears during regeneration.

Soluble factor(s) produced in injured fish optic nerve regulate the postinjury number of oligodendrocytes: Possible role of macrophages

Mammalian central nervous system (CNS) axons are virtually incapable of regenerating after injury. However, CNS neurons of lower vertebrates, such as fish and amphibians, are endowed with a high regenerative capacity. Lately, the glial cells have been credited with the regenerative ability of any specific CNS. We have previously demonstrated that many oligodendrocytes are recovered in cultures of injured rat optic nerve, while only a few oligodendrocytes are recovered from injured fish optic nerve in culture. We further demonstrated that medium conditioned by regenerating fish optic nerves (CM), which has been shown to cause axonal elongation in injured rabbit optic nerves, causes a decrease in the number of oligodendrocytes in rat glial cultures. In the present study, we demonstrate that soluble factors in the CM are capable of reducing the number of fish oligodendrocytes in fish optic nerve cultures. In addition, an inverse relationship was found between the number of macrophages and the number of oligodendrocytes. These results thus suggest that macrophages and/or activated resident microglial cells are directly or indirectly responsible for the presence of these soluble factor(s) that regulate the postinjury number of oligodendrocytes in the fish optic nerves.

New Surgical Approach To Overcome The Inability Of InjuredMammalian Axons To Grow Within Their Environment

We present a new method for creating conditions conducive to axonal growth in injured optic nerves of adult rabbits. The surgical approach consists of making a cavity in the adult rabbit optic nerve, into which a piece of nitrocellulose soaked with conditioned medium originating from regenerating fish optic nerves is implanted. In addition, daily irradiation (10 days, 5 min, 35 mW) with low energy He-Ne laser is carried out. Such a combined treatment may open a door to neurobiologists and clinicians, hoping to unravel the enigma of mammalian CNS regeneration.

Oligodendrocyte cytotoxic factor associated with fish optic nerve regeneration: implications for mammalian CNS regeneration

The limited capacity for regenerative axonal growth by adult mammalian central neurons has been attributed, at least in part, to the presence of mature oligodendrocytes, which are non-permissive for axonal growth. These cells do not interfere with growth during development, as developmental growth is largely completed before the maturation of the oligodendrocytes. Unlike mammals, fish central nervous system is endowed with a high regenerative capability. When soluble substances derived from regenerating fish optic nerves are applied to injured adult rabbit optic nerves, regenerative axonal growth is permitted. Therefore, in the present study, we tested whether the fish optic nerve, after injury, is endowed with a mechanism by which it avoids the possible inhibitory effect of the process-bearing mature oligodendrocytes. Specifically, we looked for the possible presence of soluble substances that can regulate the number of process-bearing mature oligodendrocytes. We found that soluble substances derived from regenerating fish optic nerve, when added to cultures of oligodendrocytes derived from newborn or injured adult rat optic nerves, caused a decrease in the number of process-bearing mature oligodendrocytes. Soluble substances derived from normal noninjured fish optic nerves, had a significantly lower effect. The observed decrease in the number of mature oligodendrocytes could not be mimicked by the addition of platelet-derived growth factor (PDGF), a known mitogen of oligodendrocyte progenitors which transiently inhibits their maturation. This study suggests a role to oligodendrocyte inhibitory/cytotoxic factor(s) in regeneration.

Optic Nerve Regeneration in Adult Fish and Apolipoprotein A‐I

Fish optic nerves, unlike mammalian optic nerves, are endowed with a high capacity to regenerate. Injury to fish optic nerves causes pronounced changes in the composition of pulse-labeled substances derived from the surrounding non-neuronal cells. The most prominent of these injury-induced changes is in a 28-kilodalton (kDa) polypeptide whose level increases after injury, as revealed by one-dimensional gel electrophoresis and autoradiography. The present study identified as apolipoprotein A-I (apo-A-I) a polypeptide of 28 kDa in media conditioned by regenerating fish optic nerves. The level of this polypeptide increased after injury by approximately 35%. Apo-A-I was isolated by gel-permeation chromatography from delipidated high-density lipoproteins (HDL) that had been obtained from carp plasma by sequential ultracentrifugation. Further identification of the purified protein as apo-A-I was based on its molecular mass (28 kDa) as determined by gel electrophoresis, amino acid composition, and microheterogeneity studies. The isolated protein was further analyzed by immunoblots of two-dimensional gels and was found to contain six isoforms. Western blot analysis using antibodies directed against the isolated plasma protein showed that the 28-kDa polypeptide in the preparation of soluble substances derived from the fish optic nerves (conditioned media, CM) cross-reacted immunologically with the isolated fish plasma apo-A-I. Immunoblots of two-dimensional gels revealed the presence of three apo-A-I isoforms in the CM of regenerating fish optic nerves (pIs: 6.49, 6.64, and 6.73). At least some of the apo-A-I found in the CM is derived from the nerve, as was shown by pulse labeling with [35S]methionine, followed by immunoprecipitation. The apo-A-I immunoactive polypeptides in the CM of the fish optic nerve were found in high molecular-weight, putative HDL-like particles. Immunocytochemical staining revealed that apo-A-I immunoreactive sites were present in the fish optic nerves. Higher labeling was found in injured nerves (between the site of injury and the brain) than in non-injured nerves. The accumulation of apo-A-I in nerves that are capable of regenerating may be similar to that of apo-E in sciatic nerves of mammals (a regenerative system); in contrast, although its synthesis is increased, apo-A-I does not accumulate in avian optic nerves nor does apo-E in rat optic nerves (two nonregenerative systems).

Conditioned media of regenerating fish optic nerves modulate laminin levels in glial cells.

Adult injured optic nerves of mammals show an increased distribution of laminin in the extracellular matrix after application of soluble substances in the form of conditioned media originating from growing nerves (Zak, et al., 1987). This increase could result from direct or an indirect activation of glial cells or from activation of other cellular elements. In the present study, we show that the conditioned media derived from regenerating fish optic nerves contain factors that directly modulate the level of laminin in C-6 glioma cells. The identity of the laminin was confirmed by metabolic labeling of the treated cells with [35S]methionine and by subsequent immunoprecipitation and gel electrophoresis. The level of other extracellular matrix proteins, such as fibronectin, is also increased. The significance of the presence of glial-activating substances in the conditioned media of regenerating nerves for the process of regeneration is discussed.

Ultrastructural localization of apolipoprotein A-1 in regenerating fish optic nerve

Ultrastructural localization of apolipoprotein A-1 in regenerating fish optic nerve

Morphological response of injured adult rabbit optic nerve to implants containing media conditioned by growing optic nerves

Adult rabbit retina can express regeneration-associated characteristics after optic nerve injury, provided it is supplied with appropriate diffusible substances originating from media conditioned by regenerating fish optic nerves or by optic nerves of a newborn rabbit [Hadani et al., Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 7965; Schwartz et al., Science, 228 (1985) 600]. This was shown by applying the active substances to the injured axons in the form of ‘wrap-around’ implants, consisting of collagen-coated silicone tubes which had been soaked in the conditioned media (CM). The regeneration-associated response was manifested biochemically and by sprouting of nerve fibers in culture. The present work provides morphological evidence that the implantation prolongs survival of ganglion cells and optic nerve fibers and induces new growth. Light microscopic analysis (using horseradish peroxidase (HRP) for labeling the fibers) revealed, 1 week following optic nerve injury, labeled fibers and ganglion cells in both the implanted and control (injured only or injured and implanted with collagen-coated silicone tubes free of CM) nerves. However, from the second week after the injury, distinct differences in the appearance of viable ganglion cells and labeled fibers, were seen between experimental and control preparations. In sections taken through the optic nerve, at the region distal to the site of injury, HRP-labeled fibers were seen in the experimental nerves 1 week, 2 weeks and to a significantly lesser extent 1 month after injury. In the control nerves, HRP-labeled fibers were scarcely seen from 2 weeks on after the injury. The fibers seemed to have a structure of growing tips at their terminals. Electron microscopic analysis revealed the presence of fibers with growth cones in the experimental nerves. These growth cones were found to be embedded in a scaffold-like structure formed by astrocytic processes. These results emphasize that active conditioned media implanted around the injured optic nerve of an adult rabbit causes a morphological response in addition to the already observed biochemical response.

Laminin-immunoreactive sites are induced by growth-associated triggering factors in injured rabbit optic nerve

We have previously demonstrated that application of soluble growth-associated triggering factors (GATFs) from regenerating fish optic nerve or neonatal rabbit optic nerve to a non-regenerative crushed adult rabbit optic nerve provokes regeneration-like changes in the adult rabbit. In this study we show that the responses initiated by GATFs also include a change in pattern of appearance of an extracellular matrix component, laminin, known to play a role in neurite outgrowth and elongation. These findings suggest a mechanism whereby GATFs activate the adult rabbit glial cells to produce or to accumulate laminin and thereby allow partial compensation for the low inherent regenerative ability of the adult rabbit optic nerve.

Environmental changes induced by growth-associated triggering factors in injured optic nerve of adult rabbit.

Central nervous system (CNS) neurons of mammals regenerate poorly after axonal injury. However, if an injured CNS neuron (rabbit optic nerve) is supplied with appropriate soluble substances ("growth-associated triggering factors") derived from medium conditioned by regenerating fish optic nerve or newborn rabbit optic nerve, it can express regeneration-associated characteristics. Such characteristics include a general increase in protein synthesis, changes in synthesis of specific polypeptides, and sprouting of nerve fibers in culture. The present study of rabbit optic nerves demonstrates that such active substances affect the neuronal environment (i.e., the non-neuronal cells), thereby perhaps causing a shift in the environment from an inhibitory to a regenerative supportive one. Apparently, such an environment is spontaneously achieved in injured CNS nerves of lower vertebrates (e.g., fish optic nerves), which are regenerable. Treatment of injured rabbit optic nerve with soluble factors from medium conditioned by regenerating carp optic nerve resulted in a selective increase in proliferation ([3H]thymidine incorporation) of perineural cells and the appearance of a 12-kDa polypeptide in a homogenate derived from the nerve and its associated cells. This polypeptide may be related to growth, since it comigrates in NaDodSO4/polyacrylamide gel electrophoresis with a 12-kDa polypeptide that is continuously present in a regenerative system. In addition, there were injury-induced changes in the polypeptides of the nerve that were independent of treatment with conditioned medium and were correlated with nerve maturation. The most prominent changes of this type were in 18-kDa and 25-kDa polypeptides whose levels were reduced after injury and were found to be correlated with the nerve maturation (myelination) state.

Regenerating fish optic nerves and a regeneration-like response in injured optic nerves of adult rabbits: by M. Schwartz, M. Belkin, A. Harel, et al. Science 228:600–602, 1985

Regenerating fish optic nerves and a regeneration-like response in injured optic nerves of adult rabbits: by M. Schwartz, M. Belkin, A. Harel, et al. Science 228:600–602, 1985

Regenerating fish optic nerves and a regeneration-like response in injured optic nerves of adult rabbits.

Regeneration of fish optic nerve (representing regenerative central nervous system) was accompanied by increased activity of regeneration-triggering factors produced by nonneuronal cells. A graft of regenerating fish optic nerve, or a "wrap-around" implant containing medium conditioned by it, induced a response associated with regeneration in injured optic nerves of adult rabbits (representing a nonregenerative central nervous system). This response was manifested by an increase of general protein synthesis and of selective polypeptides in the retinas and by the ability of the retina to sprout in culture.