Fish Optic Nerve Research Articles

Transglutaminase 2 Regulates HSF1 Gene Expression in the Acute Phase of Fish Optic Nerve Regeneration.

Fish retinal ganglion cells (RGCs) can regenerate after optic nerve lesions (ONLs). We previously reported that heat shock factor 1 (HSF1) and Yamanaka factors increased in the zebrafish retina 0.5-24 h after ONLs, and they led to cell survival and the transformation of neuro-stem cells. We also showed that retinoic acid (RA) signaling and transglutaminase 2 (TG2) were activated in the fish retina, performing neurite outgrowth 5-30 days after ONLs. In this study, we found that RA signaling and TG2 increased within 0.5 h in the zebrafish retina after ONLs. We examined their interaction with the TG2-specific morpholino and inhibitor due to the significantly close initiation time of TG2 and HSF1. The inhibition of TG2 led to the complete suppression of HSF1 expression. Furthermore, the results of a ChIP assay with an anti-TG2 antibody evidenced significant anti-TG2 immunoprecipitation of HSF1 genome DNA after ONLs. The inhibition of TG2 also suppressed Yamanaka factors' gene expression. This rapid increase in TG2 expression occurred 30 min after the ONLs, and RA signaling occurred 15 min before this change. The present study demonstrates that TG2 regulates Yamanaka factors via HSF1 signals in the acute phase of fish optic nerve regeneration.

Specific Activation of Yamanaka Factors via HSF1 Signaling in the Early Stage of Zebrafish Optic Nerve Regeneration.

In contrast to the case in mammals, the fish optic nerve can spontaneously regenerate and visual function can be fully restored 3-4 months after optic nerve injury (ONI). However, the regenerative mechanism behind this has remained unknown. This long process is reminiscent of the normal development of the visual system from immature neural cells to mature neurons. Here, we focused on the expression of three Yamanaka factors (Oct4, Sox2, and Klf4: OSK), which are well-known inducers of induced pluripotent stem (iPS) cells in the zebrafish retina after ONI. mRNA expression of OSK was rapidly induced in the retinal ganglion cells (RGCs) 1-3 h after ONI. Heat shock factor 1 (HSF1) mRNA was most rapidly induced in the RGCs at 0.5 h. The activation of OSK mRNA was completely suppressed by the intraocular injection of HSF1 morpholino prior to ONI. Furthermore, the chromatin immunoprecipitation assay showed the enrichment of OSK genomic DNA bound to HSF1. The present study clearly showed that the rapid activation of Yamanaka factors in the zebrafish retina was regulated by HSF1, and this sequential activation of HSF1 and OSK might provide a key to unlocking the regenerative mechanism of injured RGCs in fish.

Cultures of glial cells from optic nerve of two adult teleost fish: Astatotilapia burtoni and Danio rerio

BackgroundIn vitro studies are very useful to increase the knowledge of different cell types and could be the key to understand cell metabolism and function. Fish optic nerves (ON) can recover visual functions by reestablishing its structure and reconnecting the axons of ganglion cells. This is because fish show spontaneous regeneration of the central nervous system which does not occur in mammals. In addition, several studies have indicated that glial cells of ON have different properties in comparison to the glial cells from brain or retina. Consequently, providing an in vitro tool will be highly beneficial to increase the knowledge of these cells. New MethodWe developed a cell culture protocol to isolate glial cells from ON of two teleost fish species, Danio rerio and Astatotilapia burtoni. ResultsThe optimized protocol allowed us to obtain ON cells and brain-derived cells from adult teleost fish. These cells were characterized as glial cells and their proprieties in vitro were analyzed.Comparison with Existing Method(s): Although it is striking that ON glial cells show peculiarities, their study in vitro has been limited by the only published protocol going back to the 1990s. Our protocol makes glial cells of different fish species available for experiments and studies to increase the understanding of these glial cell types. ConclusionsThis validated and effective in vitro tool increases the possibilities on studies of glial cells from fish ON which implies a reduction in animal experimentation.

Fish-ing for neuroregenerative strategies in senescent teleosts

Fish-ing for neuroregenerative strategies in senescent teleosts

A molecular mechanism of optic nerve regeneration in fish: The retinoid signaling pathway

The fish optic nerve regeneration process takes more than 100 days after axotomy and comprises four stages: neurite sprouting (1-4 days), axonal elongation (5-30 days), synaptic refinement (35-80 days) and functional recovery (100-120 days). We screened genes specifically upregulated in each stage from axotomized fish retina. The mRNAs for heat shock protein 70 and insulin-like growth factor-1 rapidly increased in the retinal ganglion cells soon after axotomy and function as cell-survival factors. Purpurin mRNA rapidly and transiently increased in the photoreceptors and purpurin protein diffusely increased in all nuclear layers at 1-4 days after injury. The purpurin gene has an active retinol-binding site and a signal peptide. Purpurin with retinol functions as a sprouting factor for thin neurites. This neurite-sprouting effect was closely mimicked by retinoic acid and blocked by its inhibitor. We propose that purpurin works as a retinol transporter to supply retinoic acid to damaged RGCs which in turn activates target genes. We also searched for genes involved in the second stage of regeneration. The mRNA of retinoid-signaling molecules increased in retinal ganglion cells at 7-14 days after injury and tissue transglutaminase and neuronal nitric oxide synthase mRNAs, RA-target genes, increased in retinal ganglion cells at 10-30 days after injury. They function as factors for the outgrowth of thick, long neurites. Here we present a retinoid-signaling hypothesis to explain molecular events during the early stages of optic nerve regeneration in fish.

Pax2+ astrocytes in the fish optic nerve head after optic nerve crush

The transcription factor Pax2 actively participates in the development of the vertebrate visual system. In adults, Pax2 expression persists in a subpopulation of Müller cells and/or astrocytes in the retina and optic nerve head (ONH), although its function remains elusive. In a previous work we showed that the pax2 gene expression is modified and the Pax2+ astrocyte population in the ONH strongly reacted during the regeneration of the retina after a lesion in goldfish. In the present work we have analyzed Pax2 expression in the goldfish ONH after optic nerve (ON) crush. At one week post-injury, when the regenerating axons arrive at the ONH, the pax2 gene expression level increases as well as the number of Pax2+ astrocytes in this region. These Pax2+ astrocytes show a higher number of Cytokeratin (Ck)+/GFAP+ processes compared with control animals. In contrast, a different S100+ astrocyte population is not modified and persists similar to that of controls. Furthermore, we find a ring that surrounds the posterior ONH that is formed by highly reactive astrocytes, positive to Pax2, GFAP, Ck, S100, GS and ZO1. In this region we also find a source of new astrocytes Pax2+/PCNA+ that is activated after the injury. We conclude that Pax2+ astrocytes constitute a subpopulation of ONH astrocytes that strongly reacts after ON crush and supports our previous results obtained after retina regeneration. Altogether, this suggests that pax2 gene expression and Pax2+ astrocytes are probably directly involved in the process of axonal regeneration.

A Novel Look at Astrocytes

Aquaporin-4 (AQP4) water channels are located at the basolateral membrane domain of many epithelial cells involved in ion transport and secretion. These epithelial cells separate fluid compartments by forming apical tight junctions. In the brain, AQP4 is located on astrocytes in a polarized distribution: At the border to blood vessels or the pial surface, its density is very high. During ontogeny and phylogeny, astroglial cells go through a stage of expressing tight junctions, separating fluid compartments differently than in adult mammals. In adult mammals, this barrier is formed by arachnoid, choroid plexus, and endothelial cells. The ontogenetic and phylogenetic barrier transition from glial to endothelial cells correlates with the regenerative capacity of neuronal structures: Glial cells forming tight junctions, and expressing no or unpolarized AQP4 are found in the fish optic nerve and the olfactory nerve in mammals both known for their regenerative ability. It is hypothesized that highly polarized AQP4 expression and the lack of tight junctions on astrocytes increase ionic homeostasis, thus improving neuronal performance possibly at the expense of restraining neurogenesis and regeneration.

Involvement of galectins during fish optic nerve regeneration

Involvement of galectins during fish optic nerve regeneration

Growing axons in fish optic nerve are accompanied by astrocytes interconnected by tight junctions

Mammalian astrocytes are in general interconnected by gap but not by tight junctions and play an ambiguous and controversially discussed role in central nervous system regeneration. At different neuroanatomical sites, fish astrocytes are interconnected by tight junctions and desmosomes and are involved in the successful regeneration of lesioned fiber tracts. In fish, newly generated retinal ganglion cells continuously grow new axons to the optic tectum but the interrelationship between glial tight junctions and axonal growth is undefined so far. We therefore investigated the occurrence of tight junctional structures and molecules within the ribbon-shaped optic nerve of a teleost fish ( Astatotilapia burtoni) and found a predominant expression of zonula occludens protein-1 and claudin-1 in astrocytes where axons of new ganglion cells are assembled retinotopically within the optic nerve. This may support a previously formulated hypothesis according to that different properties of astrocytic membranes could be responsible for different glio-neuronal interactions which in turn may determine the micro-environmental conditions of growing axons.

Regeneration of the efferent fibers of fish optic nerve after transection

Regeneration of the efferent fibers of fish optic nerve after transection

Role of purpurin as a retinol-binding protein in goldfish retina during the early stage of optic nerve regeneration: its priming action on neurite outgrowth.

Unlike mammals, the fish optic nerve can regenerate after injury. So far, many growth or trophic factors have been shown as an axon-regenerating molecule. However, it is totally unknown what substance regulates or triggers the activity of these factors on axonal elongation. Therefore, we constructed a goldfish retina cDNA library prepared from the retina treated with optic nerve transection 5 d previously, when it was just before regrowing optic axons after injury. A cDNA clone for goldfish purpurin for which expression was upregulated during the early stage of optic nerve regeneration was isolated from the retina cDNA library. Purpurin was discovered as a secretory retinol-binding protein in developing chicken retinas. Levels of purpurin mRNA and protein transiently increased and rapidly decreased 2-5 d and 10 d after axotomy, respectively. Purpurin mRNA was localized to the photoreceptor cells, whereas the protein was diffusely found in all of the retinal layers. A recombinant purpurin alone did not affect any change of neurite outgrowth in explant culture of the control retina, whereas a concomitant addition of the recombinant purpurin and retinol first induced a drastic enhancement of neurite outgrowth. Furthermore, the action of retinol-bound purpurin was effective only in the control (untreated) retinas but not in those primed (treated) with a previous optic nerve transection. Thus, purpurin with retinol is the first candidate molecule of priming neurite outgrowth in the early stage of optic nerve regeneration in fish.

Axonal regeneration of fish optic nerve after injury.

Since Sperry's work in the 1950s, it has been known that the central nervous system (CNS) neurons of lower vertebrates such as fish and amphibians can regenerate after axotomy, whereas the CNS neurons of mammals become apoptotic after axotomy. The goldfish optic nerve (ON) is one of the most studied animal models of CNS regeneration. Morphological changes in the goldfish retina and tectum after ON transection were first researched in the 1970s-1980s. Many biochemical studies of neurite outgrowth-promoting substances were then carried out in the 1980s-1990s. Many factors have been reported to be active substances that show increased levels during fish ON regeneration, as shown by using various protein chemistry techniques. However, there are very few molecular cloning techniques for studying ON regeneration after injury. In this review article, we summarize the neurite outgrowth-promoting factors reported by other researchers and describe our strategies for searching for ON regenerating molecules using a differential hybridization technique in the goldfish visual system. The process of goldfish ON regeneration after injury is very long. It takes about half a year from the start of axonal regrowth to complete restoration of vision. The process has been classified into three stages: early, middle and late. We screened for genes with increased expression during regeneration using axotomized goldfish retinal and tectal cDNA libraries and obtained stage-specific cDNA clones that were upregulated in the retina and tectum. We further discuss functional roles of these molecules in the regeneration processes of goldfish ON.

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.

Injury‐induced gelatinase and thrombin‐like activities in regenerating and nonregenerating nervous systems

It is now widely accepted that injured nerves, like any other injured tissue, need assistance from their extracellular milieu in order to heal. We compared the postinjury activities of thrombin and gelatinases, two types of proteolytic activities known to be critically involved in tissue healing, in nonregenerative (rat optic nerve) and regenerative (fish optic nerve and rat sciatic nerve) neural tissue. Unlike gelatinases, whose induction pattern was comparable in all three nerves, thrombin-like activity differed clearly between regenerating and nonregenerating nervous systems. Postinjury levels of this latter activity seem to dictate whether it will display beneficial or detrimental effects on the capacity of the tissue for repair. The results of this study further highlight the fact that tissue repair and nerve regeneration are closely linked and that substances that are not unique to the nervous system, but participate in wound healing in general, are also crucial for regeneration or its failure in the nervous system.

Phosphorylated microtubule associated protein (MAP1B-Phos) in the retina during regeneration of the fish optic nerve.

Phosphorylated microtubule associated protein (MAP1B-Phos) in the retina during regeneration of the fish optic nerve.

Reevaluation of the growth-permissive substrate properties of goldfish optic nerve myelin and myelin proteins

To determine whether optic nerve myelin of goldfish carries mammalian-like neurite growth inhibitory proteins which can be neutralized by the antibody IN-1, myelin fractions of fish optic nerves were used as substrates for fish retinal ganglion cell axons and rat dorsal root ganglia (DRG). Axonal growth was monitored and compared with that of IN-1 treated preparations. Growth of fish retinal axons and rat DRG neurites was substantial on goldfish optic nerve myelin and no improvement was observed with IN-1. In contrast, rat CNS myelin allowed only poor growth, and number of axons and length of DRG neurites increased significantly with IN-1. In addition, proteins of fish optic nerve myelin and bovine CNS myelin were extracted, reconstituted in liposomes and applied to growth cones. When goldfish myelin proteins in liposomes were seeded onto growth cones, 77% of fish and 89% of rat DRG growth cones continued to elongate, and the proportion of elongating fish growth cones (80%) did not significantly change when liposomes were pretreated with IN-1. But 73% of fish and 93% of rat growth cones collapsed with liposomes containing proteins from bovine CNS myelin. Upon IN-1 treatment, only 24% of fish growth cones collapsed. Thus, axon growth in vitro indicates that goldfish optic nerves, which permit successful axon regeneration in vivo, lack mammalian-like neurite growth inhibitors which are neutralized by IN-1.

Macrophages, microglia, and astrocytes are rapidly activated after crush injury of the goldfish optic nerve: A light electron microscopic analysis

Several matrix and adhesion molecules in fish optic nerve, which are constitutively expressed, are increased during axonal regeneration and are primarily associated with nonneuronal cells (W.P. Battisti, Y. Shinar, M. Schwartz, P. Levitt, and M. Murray [1992] J. Neurocytol. 21:557-573). The current study examines the reactions of specific cell types to optic nerve crush and axonal regeneration. The goldfish optic nerve contains macroglia and microglia as well as a population of monocyte-derived cells (granular macrophages) unique to goldfish. Two cell types were OX-42 positive (granular macrophages and microglia), indicating monocyte lineage, each with a distinct morphology and distribution within the nerve. Within hours of the optic nerve crush, the number of OX-42-labeled cell profiles increased near the crush site, remained elevated during the time axons were elongating, and then declined. Microglia, but not granular macrophages, were phagocytically active. Astrocytes are readily identified in the normal optic nerve, but they exhibited marked morphologic changes within hours of injury, which is consistent with the contribution these cells make to the altered environment. Oligodendroglia could not be reliably identified in regenerating optic nerves until myelin was formed. A comparison of the distribution of OX-42-labeled cells with that of transforming growth factor beta-1 (TGF-beta 1) and tenascin suggests that these molecules are expressed by granular macrophages. Tenascin staining may be additionally associated with astrocytes and/or microglia. The rapid response of these nonneuronal cells to injury, their rapid phagocytic activity, and the secretion of growth-promoting factors by these cells likely contributes to the environment that supports robust regeneration by optic axons in the goldfish.

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.

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.