Fish Optic Nerve Research Articles

The enigma of myelin-associated growth inhibitors in spontaneously regenerating nervous systems

Recent results shed new light on how some nervous systems can regenerate after injury while others cannot. Until recently, it was widely believed that the main difference between systems that regenerate and those that do not lies in the normal state of their permissiveness to the regenerating axons. Thus, while nonregenerative systems, such as the rat optic nerve, were shown to contain myelin-associated growth inhibitors, regenerative systems, such as the fish optic nerve, were thought to have no such inhibitors. However, it has now been demonstrated that spontaneously regenerating systems do contain growth inhibitors, though their levels seem to be lower than in nonregenerative systems. The main difference, however, appears to reside in the system's response to injury. This article discusses the involvement of myelin-associated growth inhibitors in the spontaneously regenerating nervous system of fish, traces the apparent discrepancy, and shows how it has been resolved recently.

Chapter 27 Cytokines and cytokine-related substances regulating glial cell response to injury of the central nervous system

Publisher Summary The ability or inability of nerves to regenerate their injured axons depends on the cellular milieu surrounding the axons and its response to axonal injury. Recent research has shed more light on the nature of these cells and their associated soluble and insoluble substances in the mature nerve in the resting state and after injury. Several studies have helped to elucidate key cellular processes and substances in regeneration, and have demonstrated the involvement of regeneration-related cross-talk between the immune and the nervous systems. These findings are based on two independent lines of research, one involving studies of the fish optic nerve, and the other involving studies of mammalian sciatic nerves. Both systems regenerate readily after injury. These results suggest that macrophages and/or their products (cytokines) at the site of the injury affect local glial cells in a way that benefits regeneration. Such effects presumably include elimination of oligodendrocytes and proper activation of astrocytes. Taken together, the observations that inflammation is beneficial for regeneration and that anti-inflammatory agents promote posttraumatic rescue of fibers from secondary degeneration lead to propose that inflammation has dissimilar effects on axonal rescue and regeneration.

Fish optic nerve oligodendrocytes support axonal regeneration of fish and mammalian retinal ganglion cells

Segments from adult fish and rat retinae were explanted on myelin-marker expressing oligodendrocytes derived from the regenerating goldfish optic nerve. Fish axons grew in high density and even rat retinal axons regenerated to considerable length on the surface of the fish oligodendrocytes, suggesting that this type of fish glia has axon-growth promoting surface components that exert their influence across species boundaries. One interesting surface component of the fish oligodendrocytes as demonstrated here is the E 587 antigen, which is related to the L1 family of cell adhesion molecules. In long term cocultures of oligodendrocytes and retinal axons, the fish glial cells were found to enwrap rat axons. This suggests that the oligodendrocytes of the regenerating goldfish optic nerve/tract may, despite striking differences, represent the equivalent to mammalian optic nerve oligodendrocytes.

Axonal regeneration is associated with glial migration: Comparison between the injured optic nerves of fish and rats

The central nervous systems of mammals and fish differ significantly in their ability to regenerate. Central nervous system axons in the fish readily regenerate after injury, while in mammals they begin to elongate but their growth is aborted at the site of injury, an area previously shown to contain no glial cells. In the present study we compared the ability of glial cells to migrate and thus to repopulate the injured area in fish and rats, and used light and electron microscopy in an attempt to correlate such migration with the ability of axons to traverse this area. One week after the optic nerve was crushed, both axonal and glial responses to injury were similar in fish and rat. In both species glial cells were absent in the injured area (indicated by the disappearance of glial fibrillary acidic protein and vimentin immunoreactive cells from the site of injury in rat and fish, respectively), while at the same time axonal growth, indicated by expression of the growth-associated protein GAP-43, was restricted to the proximal part of the nerve. In fish, 2 weeks after the crush, GAP-43 staining (i.e., growing axons) was seen at the site of injury, in association with migrating vimentin-positive glial cells. One week later the site of injury in the fish optic nerve was repopulated by vimentin-positive glial cells, and GAP-43-positive axons had already traversed the site of injury and reached the distal part of the nerve.(ABSTRACT TRUNCATED AT 250 WORDS)

CDNA clones from fish optic nerve

1. 1. The present review describes the results of a cloning that was successfully employed in the study of optic nerve regeneration in fish. 2. 2. Three intermediate filaments (IFs) expressed by the glial fish optic nerve were cloned. 3. 3. By the use of this approach it was possible to resolve the controversial question of whether glial fibrillary acidic protein (GFAP) is expressed in the fish optic nerve. 4. 4. Morever, as a result of the information that emerged from the cloning, it was possible to raise well-characterized monospecific antibodies and successfully exploit them in order to determine glial cell maturation, lineage and plasticity, monitor the glial cell response to injury of the fish optic nerve, and compare it to that of a non-regenerative system.

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.

Trying to understand axonal regeneration in the CNS of fish.

In contrast to the situation in mammals and birds, neurons in the central nervous system (CNS) of fish--such as the retinal ganglion cells--are capable of regenerating their axons and restoring vision. Special properties of the glial cells and the neurons of the fish visual pathway appear to contribute to the success of axonal regeneration. The fish oligodendrocytes lack the axon growth inhibiting molecules that interfere with axonal extension in mammals. Instead, fish optic nerve oligodendrocytes support--at least in vitro--axonal elongation of fish as well as that of rat retinal axons. Moreover, the fish retinal ganglion cells re-express upon injury a set of growth-associated cell surface molecules and equip the regenerating axons throughout their path and up into their target, the tectum opticum with these molecules. This may indicate that the injured fish ganglion cells reactivate the cellular machinery necessary for axonal regrowth and pathfinding. Furthermore, the target itself provides positional marker molecules even in adult fish. These marker molecules are required to guide the regenerating axons back to their retinotopic home territory within the tectum.

Identification of an interleukin 2-like substance as a factor cytotoxic to oligodendrocytes and associated with central nervous system regeneration.

Axons of the central nervous system in adult mammals do not regenerate spontaneously after injury, partly because of the presence of oligodendrocytes that inhibit axonal growth. This is not the case in lower vertebrates (e.g., in fish), where regeneration of the optic nerve does occur spontaneously and has been correlated with the presence of factors cytotoxic to oligodendrocytes. The present study provides evidence that the substance originating from the fish optic nerves, which is cytotoxic to oligodendrocytes, is an interleukin 2-like substance.

Postinjury changes in platelet-derived growth factor-like activity in fish and rat optic nerves.

The poor regenerative ability of the CNS of mammals has been attributed, at least in part, to the presence of mature oligodendrocytes, which have been shown to inhibit axonal growth. Proliferation of oligodendrocyte progenitor cells in the rat optic nerve during development, and thereby the timing of oligodendrocyte differentiation, has been shown to depend on a factor derived from type 1 astrocytes, later characterized as platelet-derived growth factor (PDGF). In the present study we examine whether injury to the optic nerve induces changes in the levels of PDGF in spontaneously regenerating systems, compared with nonregenerating systems. Soluble substances, derived from nonneuronal cells surrounding injured fish and rat optic nerves, were prepared and examined for the presence of PDGF immunoreactivity and biological mitogenic activity on PDGF-responsive cells. The results suggest that PDGF-like mitogenic activity and immunoreactivity are present in both fish and rat optic nerves. However, in the rat optic nerve PDGF levels increased after axonal injury, whereas in the fish optic nerve injury was accompanied by an apparent decrease in PDGF-like levels. The results are discussed with respect to the possible role of PDGF in regeneration.

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.

The monoclonal antibody E587 recognizes growing (new and regenerating) retinal axons in the goldfish retinotectal pathway

E587 is a new monoclonal antibody against a 200 kDa cell-surface glycoprotein in the fish retinotectal pathway. The E587 antigen probably belongs to the class of cell adhesion molecules, and more specifically, to the family of L1-like molecules. The immunopurified protein is recognized by the antibody against the HNK1/L2 sugar epitope (associated with most cell adhesion molecules) and by a polyclonal antiserum against chick G4, which is related to the cell adhesion molecule L1 in mouse. Moreover the NH2-terminal sequence of E587 shows similarity with L1 and Ng-CAM. The E587 immunostaining pattern in the fish retinotectal pathway suggests that the E587 antigen is a growth-associated molecule on fish retinal axons. In fish embryos, all retinal axons are labeled. In adult fish, however, only the young axons from newly added ganglion cells carry E587 staining. After optic nerve transection (ONS) and retinal axonal regeneration, all axons reexpress the E587 antigen into their terminal processes in the tectal retinorecipient layers. The reexpression of the E587 antigen is temporally regulated, and E587 immunoreactivity declines by 7 months and disappears at 12 months after ONS. We hypothesize that the E587 antigen may mediate axon-axon associations. In its restricted appearance on young axons in normal adult fish, it may contribute to the selective fasciculation of the newest axons with young axons and thus participate in the creation of the age-related fiber organization in the fish optic nerve.

Isolation and sequence analysis of two intermediate filament cDNA clones from fish optic nerve

The high post-traumatic regenerative ability of fish central nervous system has been partially attributed to the hospitable nature of the surrounding non-neuronal cells and their appropriate response to injury. Uncovering the correlation between fish non-neuronal cell structure and behavior might yield a better understanding of what makes them supportive to axonal growth. Towards this goal, structural proteins expressed by fish non-neuronal cells need to be characterized. In the present study we isolated cDNA clones encoding fish intermediate filaments which are prominent structural proteins in astrocytes. Among the isolated clones, one was identified as fish vimentin and another was found identical to the cloned fish keratin 8. Results are discussed with respect to the use of these cDNAs for further understanding of fish non-neuronal cell plasticity.

The design of the optic nerve in fish

Fish have large eyes, with short optic nerves that are continually flexed by compensatory eye movements during swimming. Here, I review the tissue construction of the fish optic nerve, to see how the glia and axons are adapted to withstand these mechanical stresses, which are not normally encountered by CNS tissue within the skull. As in other lower vertebrates, the optic nerve astrocytes are highly unusual: their intermediate filaments are composed of cytokeratins (Giordano et al., 1989), not GFAP. Their processes are linked together by desmosomes, forming thin transverse lace-like partitions, placed at quasi-regular intervals longitudinally (Maggs & Scholes, 1990). This accordion-like arrangement is interpreted as providing a flexible tissue-skeleton for the optic nerve. A new observation is that the optic axons run in coherent parallel waves. This pattern, which is complementary to that of the astroglia, reversibly accommodates limited axial stretches. The waves are equivalent to those underlying the optical banding of Fontana (1781) in peripheral nerves, but wavelength (30 microns) and amplitude (5 microns) are about an order of magnitude less, reflecting the much smaller average size of the optic axons. The pattern also occurs in mammals, and may be restricted to the visual pathway: if present elsewhere in the CNS, nerve-fiber waves are inconspicuous at best. In fish, the astroglial partitions occur in register with the waves, suggesting that steric interactions between developing axons and glia may help to establish, or stabilize, the regular longitudinal spacing. This may have functional as well as mechanical implications, since the astrocytes form perinodal associations and their pattern is one which strongly clusters the nodes of Ranvier.

Growth of regenerating goldfish axons is inhibited by rat oligodendrocytes and CNS myelin but not but not by goldfish optic nerve tract oligodendrocytelike cells and fish CNS myelin

Encounters of regenerating goldfish retinal axons with oligodendrocytes and CNS myelin of mammals and fish were monitored in in vitro assays. Upon contact with highly branched rat oligodendrocytes, goldfish axons collapsed or grew around but never crossed these cells. However, in the presence of the antibody IN-1 against the oligodendrocyte-associated growth-inhibitory proteins, axons did grow over highly branched oligodencrocytes. In contrast to the mammalian oligodendrocytes, goldfish optic nerve/tract-derived oligodendrocytelike cells allowed the growth of axons across their surface and even along their processes. The fish growth cones avoided entering the region of rat CNS myelin applied to polylysine/laminin-coated coverslips or failed to elongate on this substrate. They were, however, able to pass over CNS myelin of fish. When exposed to rat CNS myelin as the sole substrate, axonal outgrowth from fish retinal explants was inhibited almost entirely. However, outgrowth on fish CNS myelin was substantial, but many more axons extended on fish or rat brain membranes that were depleted of myelin. Thus, goldfish retinal axons are sensitive to the axon-growth-inhibiting cell-surface molecules of mammalian oligodendrocytes as well as CNS myelin. Fish optic nerve oligodendrocytelike cells and fish CNS myelin lack these inhibitory properties and are growth permissive. These in vitro experiments suggest that the success of axonal regeneration in the fish optic nerve is causally related to the presence of growth-permissive properties and to the absence of growth inhibitors on fish optic nerve/tract oligodendrocytelike cells.

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.

Diversity amongst the microglia in growing and regenerating fish CNS: Immunohistochemical characterization using FL. 1, an anti‐macrophage monoclonal antibody

We have immunohistochemically characterized the forms and distribution of microglia--the macrophages of the CNS--in fish, using a new monoclonal antibody (mAb), FL.1. This mAb specifically reacts with resident macrophages throughout the body in Oreochromine fish, including Kuppfer cells, gut-associated myeloid cells, and peritoneal macrophages, as well as with microglia, but circulating monocytes are not labelled with FL.1. The FL.1-epitope, which is lost following treatment with reducing agents, has an extracellular location and is associated with three integral membrane glycoprotein variants. FL.1-staining shows that microglia are extremely abundant throughout the fish CNS. For example, they comprise a third of the glia in the optic nerve, and 30% of all cells, including neurons, in the spinal cord, i.e., fish have about tenfold more microglia than mammals. Two forms of FL.1-positive microglia are predominant in fish, one resembling their mammalian counterparts, but less ramified, and the other comprising smaller rounded cells with very little cytoplasm, which are most numerous in the ependymal region of the optic tectum. Apart from the conventional microglia, the optic nerves also contain large lipid-laden macrophages which comprise a third form of FL.1-positive cell in the CNS. Fish optic nerves contain astrocytes of a distinct type which form reticular networks, but lack connections to capillaries (Maggs and Scholes, J. Neurosci. 1990;10:1600-1614). The co-distribution of foamy macrophages may have a metabolic role that is performed by ordinary astrocytes elsewhere in the CNS. An antiserum against the beta 2 subunit of the human leukocyte integrins (Kishimoto et al., Cell 1987a; 50:193-202) was found selectively to recognize the foamy macrophages in Oreochromis. Following lesion to the optic nerve, FL.1-labelling shows that microglia proliferate throughout the visual pathway. In the optic tectum, the additional FL.1-positive cells are concentrated in the vicinity of degenerating retinal axons and their terminals. Most of the microglia in the injured optic nerve have amoeboid morphologies, and the foamy macrophages become depleted.

Glial fibrillary acidic protein in the fish optic nerve

The intermediate filament glial fibrillary acidic protein (GFAP) is the predominant cytoskeletal protein of mature glial cells in the mammalian nervous system. The nervous systems of lower vertebrates, such as fish, have been examined for the presence of GFAP and several investigators have shown that goldfish (Carassius auratus) brain contains GFAP-positive astrocytes. The same studies have demonstrated that, in contrast to the brain, the optic nerve of goldfish did not show any GFAP immunoreactivity, suggesting that this intermediate filament protein is not expressed in fish optic nerve astrocytes. The present study shows, however, that the monoclonal antibodies to porcine GFAP react with the optic nerve of carp (Cyprinus carpio), another member of the goldfish family. These antibodies to porcine GFAP cross react with rat brain and carp optic nerve, yielding a band of approximately 52 kDa in both species. Northern blot analysis using mouse GFAP DNA probe revealed that carp optic nerve RNA contains two transcripts of 2.3 and 2.1 kb, which hybridize with the mouse GFAP probe. Injury to the carp optic nerve was followed by a decrease of GFAP immunoreactivity from neural tissue and a strong expression around blood vessels and connective tissues. On the basis of these observations and within the limitation of the techniques it is reasonable to conclude that the carp optic nerve expresses GFAP immunoreactivity and that the pattern of expression of this intermediate filament protein is altered after injury. Such an alteration might be relevant to the process of regeneration.

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.

Immunological evidence that the neural adhesion molecule L1 is expressed in fish brain and optic nerve: possible association with optic nerve regeneration

In the mammalian peripheral nervous system (PNS), expression of the neural adhesion molecule L1 on Schwann cells and neurons has been correlated with axonal growth during development and regeneration. The present study was undertaken to examine whether a similar correlation exists between a lesion-induced increase of L1 expression and regenerative capacity in the central nervous system (CNS). The fish optic nerve was used as a model for a succesfully regenerating region of the CNS. Immunochemical and immunohistological experiments carried out with immunoaffinity purified polyclonal antibodies, generated against L1 from mouse brain, showed that carp optic nerve and brain, but not liver, contained L1 immunoreactivity. Western blot analysis of brain tissue yielded one distinct band at 200 kDa, while a double band at 200 kDa and two low-molecular weight bands at 120 and 100 kDa, possibly degradation products, were seen in the optic nerve. Immunohistological examination of normal optic nerves revealed L1 immunoreactivity, predominantly associated with connective tissue boundaries of nerve fascicles and with blood vessels, as well as inside axonal fascicles. L1 immunoreactivity was increased by 25%, 8 days after crushing of the optic nerve, as determined by radioimmunoassay on a nerve segment distal to the site of injury and compared with untreated control nerves. Increased levels of L1 were also seen by immunohistology and found to be predominantly associated, as in the normal nerve, with connective tissue boundaries and blood vessels. These observations suggest that a lesion-induced increase in L1 expression in the fish optic nerve is associated with axonal regrowth in the CNS.