Glial cell types, lineages, and response to injury in rat and fish: implications for regeneration.

Axons of the mammalian central nervous system do not regenerate spontaneously after axonal injury, unlike the central nervous system axons of fish and amphibians and the peripheral nervous system of mammals, which possess a good regenerative ability (Grafstein: The Retina: A Model for Cell Biology Studies, Part II, 1986; Kiernan: Biol Rev 54:155-197, 1979; Murray: J Comp Neurol 168:175-196, 1976; Ramón y Cajal: Degeneration and Regeneration of the Nervous System, 1928; Reier and Webster: J Neurocytol 3:591-618, 1974; Sperry: Physiol Zool 23:351-361, 1948). It was previously believed that intrinsic differences between the central nervous system neurons of mammals and fish account for their differences in regenerative ability. The past decade, however, has seen an accumulation of evidence, indicating that mammalian central nervous system neurons are able to regenerate injured axons, at least to some extent. This was first demonstrated by Aguayo and colleagues (David and Aguayo: Science 214:931-933, 1981; Kierstead et al: Science 246:255-257, 1989), who showed that injured mammalian central nervous system axons can grow for a considerable distance into an autograft of a peripheral nerve. It was also demonstrated that injured rabbit optic axons can regenerate into their own environment (i.e., into the distal part of the injured optic nerve), if the injured nerve is treated so as to make it conducive for growth (Lavie et al: J Comp Neurol 298:293-314, 1990; Eitan et al: Science 264:1764-1768, 1994).(ABSTRACT TRUNCATED AT 250 WORDS)

Nonpermissive nature of fish optic nerves to axonal growth is due to presence of myelin-associated growth inhibitors.

Fish optic nerve sections were recently shown to be nonpermissive to growth of adult retinal axons. In addition, fish optic nerve myelin was found to inhibit growth of adult retinal axons and this inhibition was neutralized by IN-1 antibodies (known to block rat myelin-associated inhibitors). In this study we examined whether the growth nonpermissiveness of fish optic nerves which had not been injured prior to their excision results, at least in part, from the presence of myelin-associated growth inhibitors. It was found that preincubation of the sections with IN-1 antibodies, known to recognize and neutralize the myelin-associated growth inhibitors of the rat central nervous system, increases sixfold the number of axons that grow on these sections. This demonstrates that fish myelin-associated growth inhibitors, which are similar to those of rat, are at least partly responsible for the growth nonpermissiveness of normal fish optic nerves.

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.

Presence of growth inhibitors in fish optic nerve myelin: postinjury changes.

This study shows that the fish optic nerve, which is able to regenerate after injury, contains myelin-associated growth inhibitors similar to the growth inhibitors present in mammalian central nervous system (CNS) myelin. The ability of nerves to regenerate was previously correlated with the ability of sections from these nerves to support neuronal attachment and axonal growth in vitro. Thus neuroblastoma cells or embryonic neurons became attached to and grew axons on sections of rat sciatic nerve or fish optic nerve, which are spontaneously regenerating systems, but not on sections of rat optic nerve, a nonregenerating system. Failure of the latter to support axonal growth has been attributed, at least in part, to growth inhibitors. Recently it was shown that adult neurons, which differ in their growth requirement from embryonic neurons, are unable to extend neurites on sections of normal sciatic nerve but are able to extend neurites on sections of sciatic nerve that was injured prior to its excision. We found a similar situation in the fish optic nerve, i.e., that the nerve is normally not permissive to growth of adult retinal axons but becomes growth permissive after injury. The nonpermissiveness of the normal fish optic nerve was found to correlate with the presence of myelin-associated growth-inhibitory molecules. This inhibitory activity of fish myelin was neutralized by IN-1 antibodies, known to neutralize rat myelin growth inhibitors. The results thus demonstrate that fish optic nerve myelin contains inhibitors apparently similar or even identical to those of rat, but possibly present in lower amounts than in the rat. Results are discussed with respect to the possibility that fish optic nerve, like the rat sciatic nerve and unlike the rat optic nerve, undergoes certain changes after injury that support regeneration of adult neurons. Such changes might include elimination or neutralization of growth inhibitors.

Vimentin immunoreactive glial cells in the fish optic nerve: implications for regeneration.

The poor regenerative ability of neurons of the central nervous system in mammals, as compared with their counterpart in fish or amphibians, is thought to stem from differences in their immediate nonneuronal environment and its response to axonal injury. We describe one aspect of the environmental response to axonal injury in a spontaneously regenerating system--the fish optic nerve. The aspect under investigation was the reaction of glial cells at the injury site. This was examined by the use of antibodies that specifically recognize vimentin in fish glial cells. In the present study, affinity-purified vimentin antibodies were raised against a nonconserved N-terminal 14-amino acid peptide, which was predicted from the nucleotide sequence of vimentin. These antibodies were found to react specifically with glial cells in vitro. Moreover, the antivimentin antibodies stained both the optic nerve and the optic tract, but with different patterns. Specificity of the antibodies was verified by protein immunoblotting, tissue distribution, and labeling patterns. After injury, vimentin immunoreactivity initially disappeared from the site of the lesion due to cell death. Early signs of glial cell migration toward the injury site were evident a few days later. It is suggested that the reappearance of vimentin-positive glial cells at the site of injury is associated with axonal elongation across it, and that they contribute to the regenerative ability of the fish optic nerve.

Astrocytes play a major role in the control of neuronal proliferation in vitro.

The elements that control neuronal proliferation are largely unknown. Proliferating neurons in cultures of goldfish brain were studied in an attempt to identify the cell types involved. Neuronal proliferation was found to occur only when the neuronal stem cells were in direct contact with astrocytes, and never directly on the substrate. The regulation of neuronal proliferation thus appears to be mediated, at least in part, by contact with astrocytes. In addition, neurite extension was inhibited by medium conditioned by fish astrocytes. Since neurite extension and neuronal proliferation are mutually exclusive processes, inhibition of neurite extension by soluble substances derived from the astrocytes is probably one of the mechanisms controlling neuronal proliferation. The complex reciprocal relationship between neurons and astrocytes is also demonstrated by an observed inhibition of astrocytic proliferation by medium conditioned by differentiating fish neurons. This inhibition of astrocytic proliferation might be part of a mechanism through which interference with neuronal differentiation by astrocytes is avoided. The results of this study thus suggest that astrocytes, in addition to their known roles in controlling neuronal migration, neuronal differentiation and neurite elongation, may also play a role in the control of neuronal proliferation.

Characteristics of fish glial cells in culture: possible implications as to their lineage.

Regeneration of injured central nervous system axons is largely dependent on the response of the associated nonneuronal glial cells to injury. Glial cells of the mammalian central nervous system, unlike those of fish, are apparently not conducive to axonal regeneration. While the lineage of rat glial cells is well characterized and its role in the support or inhibition of regenerative growth is beginning to be understood, little is known about fish glial cells. Accordingly, glial cells in cultures of adult goldfish brain and of newly hatched goldfish larvae were studied in an attempt to establish their lineage. The cells were identified by means of indirect immunofluorescence, using antibodies against fish astrocytes and oligodendrocytes. The cell count in the cultures increased from a small number of cells at 24 h after plating to a large number of both astrocytes and oligodendrocytes after 1 week in culture. Both of these cell types had originated from proliferating cells, as shown by their uptake of tritiated thymidine and by the inhibition of cell proliferation by 5-fluoro-2'-deoxyuridine. Both astrocytes, i.e., glial fibrillary acidic protein-positive cells, and oligodendrocytes, i.e., 6D2-positive cells, were positively labeled also by A2B5 antibodies, which are known to label progenitors of type-2 astrocytes and oligodendrocytes in the rat optic nerve. The results suggest that A2B5 positive progenitor cells in the goldfish central nervous system, as in the rat optic nerve, might be a common progenitor of astrocytes and oligodendrocytes.

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.

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.

Glial response to axonal injury: in vitro manifestation and implication for regeneration.

Crushed fish optic axons readily regenerate, while similarly injured rat optic axons do not; the reasons for the differences in regeneration ability may lie in differences in the environment of the axons. We have cultured glial cells from previously crushed optic nerves of fish and rat to determine whether a relationship exists between the ability to regenerate and the nature of the responses of the associated nonneuronal cells to injury. The glial cells were examined using indirect immunofluorescence with antibodies to known glial markers. In the rat cultures, mature GalC oligodendrocytes, which are known to be nonpermissive for axonal growth, were abundant. In contrast, in the fish cultures mature oligodendrocytes were rare, but A2B5 positive cells were abundant. The high number of A2B5 positive cells in the fish may suggest a high number of immature cells. This interpretation, however, should wait until evidence for glial cell lineage of the fish is available. Additional indication is provided also in the present study that the number of mature oligodendrocytes in the fish is regulated by elements external to the nerve. This study thus demonstrates an important difference between rat and fish optic nerves in the response of glial cells to the optic nerve injury.