Explant culture of adult goldfish retina: A model for the study of CNS regeneration

doi:10.1016/0006-8993(79)90194-X

Brain Research

Volume 161, Issue 1, 26 January 1979, Pages 39-53

https://doi.org/10.1016/0006-8993(79)90194-X Get rights and content

Abstract

Conditions are described for culture of retinal explants of adult goldfish which favour outgrowth of neuritic processes onto a substratum. A growth index to quantitate the outgrowth was developed. If the optic nerve is crushed several days prior to explanation, a marked enhancement of neuritic outgrowth is seen relative to control retinas. Histological examination of the explants revealed that retinal ganglion cells in explants from unoperated eyes became hypertrophied in vitro with a time course similar to that observed in vivo following optic nerve crush. Experiments with hemiaxotomized retinas indicate that the perikaryal regenerative response is mediated intracellularly.

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Citation Excerpt :
The retina is an attractive in vitro model system because the tissue can be easily obtained and cultured (Bähr et al., 1988; Landreth and Agranoff, 1976; Halfter et al., 1983). Furthermore, retinal explants can yield tissue slices of relatively uniform thickness that survive well in culture and produce quantifiable neurite outgrowth (Landreth and Agranoff, 1979). Early work with retinal tissue was focused on exploring the role of molecular gradients in modulating CNS axon growth (Turner et al., 1982; Snow et al., 1991; Gottlieb et al., 1976).
A variety of in vitro models have been developed to understand the mechanisms underlying the regenerative failure of central nervous system (CNS) axons, and to guide pre-clinical development of regeneration-promoting therapeutics. These range from single-cell based assays that typically focus on molecular mechanisms to organotypic assays that aim to recapitulate in vivo behavior. By utilizing a combination of models, researchers can balance the speed, convenience, and mechanistic resolution of simpler models with the biological relevance of more complex models. This review will discuss a number of models that have been used to build our understanding of the molecular mechanisms of CNS axon regeneration.
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Like most central nervous system pathways in mature mammals, the optic nerve cannot regenerate when injured, leaving victims of traumatic nerve damage or degenerative diseases such as glaucoma with life-long visual disabilities. This situation can be partially reversed by activating the intrinsic growth state of retinal ganglion cells (RGCs), the projection neurons of the retina. Intraocular inflammation causes infiltrative cells of the innate immune system to release a recently discovered growth factor, oncomodulin (Ocm). Ocm binds to a high-affinity receptor on RGCs in a cAMP-dependent fashion and stimulates appreciable regeneration. A complementary way to stimulate regeneration is by deleting the gene for PTEN phosphatase, a suppressor of the phosphatidyl inositol-3 kinase-Akt cell-signaling pathway. Combining intraocular inflammation, cAMP, and pten gene deletion has a strong synergistic effect, enabling RGCs to regenerate injured axons the full length of the optic nerve and into the appropriate central target areas, where they form synapses and help restore simple visual responses. However, the most RGCs remain unable to regenerate their axons, and it is important to understand the reasons for this. Unlike mammals, fish and amphibians can regenerate their optic nerves and recover normal vision throughout life, and a great deal of research has been directed toward understanding the molecular bases of this phenomenon. It is likely that insights from studies in lower vertebrates will enable us to enhance optic nerve regeneration in mammals.
Neurites outgrowth and amino acids levels in goldfish retina under hypo-osmotic or hyper-osmotic conditions
2012, International Journal of Developmental Neuroscience
Citation Excerpt :
The total number of animals per experiment was 12–18, 3 for each condition. The nutrient medium was Leibovitz, L-15 (free of taurine), 3 ml per dish (Sigma) with 0.1 mg/ml of gentamicin and 20 mM of (N-2-hydroethyl]piperazine-N′-2-ethanesulfonic acid) (HEPES) (Landreth and Agranoff, 1979; Lima et al., 1988, 1989a). Hypo-osmotic media were done by dilution of 10% with distilled water at 24 and 72 h in culture (Cubillán et al., 2009).
Amino acids are known to play relevant roles as osmolytes in various tissues, including the retina. Taurine is one of these active molecules. In addition, taurine stimulates outgrowth from the goldfish retina by mechanisms that include extracellular matrix, calcium fluxes and protein phosphorylation. The present report aims to explore the effect of medium osmolarity on goldfish retinal outgrowth and the possible modifications produced by changing eye osmolarity on amino acid levels in the retina. Goldfish retinal explants were obtained 10 days after crush of the optic nerve and cultured under iso-, hypo- or hyper-osmotic conditions. Hypo-osmotic medium was prepared by diluting the solutions 10% twice, preserving fetal calf serum concentration. Hyper-osmotic medium was done by adding 50 or 100 mM urea or mannitol. Evaluation of length and density of neurites was performed 5 days after plating. Outgrowth was reduced in hypo- and in hyper-osmotic conditions. Taurine, 4 mM, increased length and density of neurites in iso-osmotic, and produced stimulatory effects under both hyper-osmotic conditions. The in vivo modification of osmolarity by intraocular injection of water or 100 mM urea modified levels of free amino acids in the retina. Taurine and aspartate retinal levels increased in a time-dependent manner after hypo- and hyper-osmotic solution injections. Serine, threonine, arginine, γ-aminobutyric acid, alanine and tyrosine were elevated in hyper-osmotic conditions. Outgrowth in vitro, after in vivo osmolarity changes, was higher in the absence of taurine, but did not increase in the presence of the amino acid. The fact that certain outgrowth took place in these conditions support that the impairment was not due to tissue damage. Rather, the effects might be related to the cascade of kinase events described during osmolarity variations. The time course under these conditions produced adjustments in ganglion cells probably related to taurine transporter, and phosphorylation of specific proteins.
Gene expression analysis of zebrafish retinal ganglion cells during optic nerve regeneration identifies KLF6a and KLF7a as important regulators of axon regeneration
2007, Developmental Biology
Unlike mammals, teleost fish are able to mount an efficient and robust regenerative response following optic nerve injury. Although it is clear that changes in gene expression accompany axonal regeneration, the extent of this genomic response is not known. To identify genes involved in successful nerve regeneration, we analyzed gene expression in zebrafish retinal ganglion cells (RGCs) regenerating their axons following optic nerve injury. Microarray analysis of RNA isolated by laser capture microdissection from uninjured and 3-day post-optic nerve injured RGCs identified 347 up-regulated and 29 down-regulated genes. Quantitative RT-PCR and in situ hybridization were used to verify the change in expression of 19 genes in this set. Gene ontological analysis of the data set suggests regenerating neurons up-regulate genes associated with RGC development. However, not all regeneration-associated genes are expressed in differentiating RGCs indicating the regeneration is not simply a recapitulation of development. Knockdown of six highly induced regeneration-associated genes identified two, KLF6a and KLF7a, that together were necessary for robust RGC axon re-growth. These results implicate KLF6a and KLF7a as important mediators of optic nerve regeneration and suggest that not all induced genes are essential to mount a regenerative response.
Medium requirements for neuritic outgrowth from goldfish retinal explants and the trophic effect of taurine
2002, International Journal of Developmental Neuroscience
Citation Excerpt :
Outgrowth of neurites from goldfish retinal explants occurs after lesion of the optic nerve (Lima et al., 1988). Conditions of the culture include the addition of fetal calf serum (Landreth and Agranoff, 1979), since in the absence of it neuritic outgrowth is dramatically restricted, even in the presence of trophic molecules, such as taurine (Lima et al., 1988). There have been several approaches to solve the important issue of studying the influence of stimulating agents per se without the concomitant effect of several substances present in the serum (Caffe et al., 2001; Espinosa de los Monteros et al., 1997; Sholl-Franco and Araujo, 1997; Winkler et al., 2000).
The use of culture media of known composition are necessary for studying the role of trophic molecules. Since most of the in vitro research on regeneration of the optic nerve has been performed in the presence of fetal calf serum, the aim of this study was to obtain a medium in which the neuritic outgrowth from post-crush goldfish retinal explants could take place without adding fetal calf serum. After the lesion of the optic nerve (10 days), the retina of goldfish was dissected and explants were cultured for 5 and 10 days in the absence or in the presence of fetal calf serum, at which time the neuritic outgrowth was determined. Various concentrations and combinations of glucose, albumin, calcium, HEPES and taurine were used. The highest neuritic outgrowth was observed in the presence of fetal calf serum, in which condition the amino acid taurine increased length and density of neurites. Media supplemented with albumin, calcium or HEPES did not modify the outgrowth of neurites from the explants. However, glucose favored the neuritic outgrowth in a bell-shaped manner, although fibers were thinner than those observed in the presence of fetal calf serum. Taurine did not stimulate outgrowth of neurites from explants growing in a medium with optimal concentrations of glucose, indicating that elements of the fetal calf serum are determinant for the trophic effect of taurine. The present results contribute to further studies, such as those related to the effect of taurine and of trophic factors derived from the optic tectum, which would be performed in the presence of a medium free of fetal calf serum.
A vulnerable period of colchicine toxicity during goldfish optic nerve regeneration
1999, Brain Research
The effects of intraocular (i.o.) administration of the alkaloid colchicine on visual recovery following axotomy of the goldfish optic nerve were investigated. Under the experimental conditions used, control goldfish recovered vision, measured behaviorally, within 5–7 weeks of retro-orbital optic nerve crush. Fish injected i.o. with 0.1 μg of colchicine within 3 days of optic nerve crush (post-crush; PC) recovered vision after some delay relative to control fish, while injection with colchicine between 7 and 14 days PC produced a much more profound inhibition of recovery of vision, in most cases a complete block for the duration of the study (98 days). Further evidence for a delayed susceptibility of the regenerating optic nerve to colchicine following crush was reflected in a suppression of neurite outgrowth normally seen in explanted retinal tissue taken from PC goldfish. In addition, retrograde transport of the fluorescent dye 4-(4-didecylaminostyryl)-N-methylpyridinium iodide from the optic tectum to the retina as a measure of axonal continuity revealed substantially less labeling following i.o. administration of colchicine 1 week PC when compared to retinas from fish receiving colchicine at the time of optic nerve crush. Histological sections of the retina showed no evidence of residual retinal damage resulting from the colchicine injections or from interactions of axotomy and the drug administration. These results indicate a period of increased vulnerability of the regenerating visual system to the toxic effects of i.o. administered colchicine, beginning 3–5 days PC, and remaining until regenerating optic nerve fibers have begun to reach the tectum. While colchicine has many known effects on nerve function, it is proposed that the delayed susceptibility to disruption of regeneration observed in these experiments is largely, if not entirely, attributable to a colchicine-induced accumulation of tubulin heterodimers, which are known to block microtubule assembly and to participate in a feedback inhibition of tubulin synthesis. Thus, it is during the maximal induction of tubulin synthesis and of microtubule formation which normally occurs several days following axotomy that colchicine has its greatest effect. The results suggest that colchicine may be especially neurotoxic during neural development and regeneration.

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Explant culture of adult goldfish retina: A model for the study of CNS regeneration

Abstract

Brain Research

Exp. Neurol.

Brain Research

Exp. Neurol.

Exp. Neurol.

Exp. Neurol.

Brain Research

Brain Research

Develop. Biol.

Exp. Neurol.

Brain Research

Brain Research

Exp. Neurol.

Brain Research

Brain Research

Changes in RNA metabolism in response to axonomy

Reconstituted rat-tail collagen used as substrate for tissue culture on coverslips in Maximow slides and roller tubes

Lab. Invest.

Poly(A)-RNA in goldfisk retina and optic nerve regeneration

RNA metabolism and optic nerve regeneration in the goldfish

J. Neurochem.

La presence d'un tissu antagoniste maintient la differenciation d'un tissu cultive en dehors de l'organisme

C.R. Soc. Biol. (Paris)

Quelques resultats de la method de culture des tissues. IV. La retine

Arch. Zool. Exp. Gen.

Neuron culture from adult goldfish

J. Neurobiol.

Plaque formation and isolation of pure lines with poliomyelitis viruses

J. exp. Med.

The growth of cells on a transparent gel of reconstituted rat tail collagen

J. nat. Cancer Inst.