Retinal Pieces Research Articles

Regeneration of optic nerve fibers with unoprostone, a prostaglandin-related antiglaucoma drug, in adult cats

We investigated the effects of unoprostone on neurite extension of cultured retinal pieces and axonal regeneration of retinal ganglion cells in the crushed optic nerve of adult cats. The retinal pieces were cultured with unoprostone or its primary metabolite, M1, dissolved in DMSO or polysorbate for 14 days, and the number and length of Tau-1-positive neurites and glial processes labeled with anti-glial fibrillary acidic protein antibodies were examined. After the optic nerve was crushed, unoprostone was injected into the vitreous body and the crushed site. On day 12, wheat germ agglutinin-conjugated horseradish peroxidase was injected into the vitreous body to anterogradely label the regenerated axons. On day 14, the optic nerve was excised and longitudinally sectioned. After peroxidase reaction, the number of axons regenerating beyond the crush site was examined. The greatest number of neurites protruded from the cultured retinal pieces in 3 μM unoprostone and 3 μM M1. The neurite length was also the longest at 3 μM unoprostone and 3 μM M1, in which no glial processes were detected. After injections of 3 μM unoprostone, the final concentration in the vitreous humor, into the vitreous body and the crush site, the optic nerve fibers regenerated and extended beyond the crush site. In contrast, almost no fibers extended beyond the crush site after injection of phosphate-buffered saline. The results indicate that intravitreal injection of unoprostone promotes regeneration of crushed optic nerve fibers in adult cats.

Modification of culture method of retinal vascular endothelial cells in vitro

Background The in vitro culture of retinal vascular endothelial cells is the foundation of experimental study of retinal vascular disease. Shortage of human donor eyeballs is a main limiting for the laboratory work. The culture method of rat-derived vascular endothelial cells has been established. However, this method is not enough effective because of severer cellullar injury. Objective Present study was to establish a simple and high effective method for the culture of vascular endothelial cells in vitro. Methods The retinas from 5 SPF SD rats was digested by 0. 1% collagenase and cultured with explant culture method. 20% fetal bovine serum, vascular endothelial growth factor ( VEGF) , insulin-transferrin-selenium( ITS) were composed into the endothelial cell culture medium, and enough blowing was performed to get the cells and fragments from retinal tissue. The cellular suspension was prepared and cultured consequently on human fibronectin-coated culture flasks. Cultured vascular endothelial cells were identified by anti-von Willebrand staining factor. Results The cells emerged from the tissue mass,and cells and some tissue fragments attached to the wall after 24 hours of seeding. The cells grew to show the fusiform in 4 days and merged together in 5 to 6 days,and a cell monolayer was seen in the 14th day after culture. The endothelial cells showed the positive response for von Willebrand factor. After passage, the merging-growth statue of the cells was regained in 2 hours after culture. Conclusion Use of retinal pieces and collagenase-digestion can get the vascular endothelial cells with better activity in vitro. The culture method based on highly selective endothelial cell culture medium associated to FN adhesion-promoting is helpful for gaining the purified of endothelial cells. Key words: Retina; Vascular endothelial cells; Culture in vitro

α2-Adrenergic Receptors and Their Core Involvement in the Process of Axonal Growth in Retinal Explants

To determine the patterns of α2-adrenergic receptor (α2-AR) subtype expression in normal and degenerated retinas and to analyze the response of these receptors to the α2-AR agonist brimonidine tartrate (BT). The binding characteristics of α2-ARs in the retina were evaluated in experimental and matching sham groups by in vitro quantitative autoradiographic saturation with [(3)H]-clonidine. Retinal explants from juvenile and adult rats with either elevated intraocular pressure or after optic nerve crush (ONC) were cultured with BT over 96 hours in vitro to analyze the effects of BT on axonal growth by videomicroscopy and axon counting. Changes in retinal protein expression by BT were monitored by two-dimensional gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The total number of α2-ARs in the retina increased significantly after ONC compared with the sham group. BT supported axonal growth in the juvenile, glaucomatous, and injured retinas (P < 0.004) most effectively at a concentration of 0.001 mg/mL, without influencing the axonal growth rate. Immediate supplementation of BT was more effective than delayed supplementation (P < 0.001). Proteomic analysis revealed treatment-specific expression patterns of glial fibrillary acidic protein (GFAP), glucose-related protein (GRP)58, platelet-activating factor (PAF), and laminin-binding protein (LBP). These data are the first to show differences in α2-AR expression in normal and degenerated retinas. BT supports neuronal growth in cultured retinal pieces, suggesting that α2-ARs play a role in retinal metabolism.

A novel ROCK inhibitor, Y-39983, promotes regeneration of crushed axons of retinal ganglion cells into the optic nerve of adult cats

We investigated the effect of a novel ROCK inhibitor, Y-39983, on neurite regeneration in vitro and axonal regeneration in the crushed cat optic nerve in vivo. To determine the effective dose for neurite regeneration, retinal pieces were cultured with ROCK inhibitors, Y-39983 or Y-27632, a well-characterized ROCK inhibitor, and the number and length of TUJ-1-positive neurites were evaluated. The greatest number of neurites protruded at a dose of 3–10 μM Y-39983 and at a dose of 10–100 μM Y-27632, respectively. The neurite number at maximum effect of Y-39983 was greater than that of Y-27632. No significant difference was observed between values of neurite length with the inhibitors. Based on this finding, we examined the effect of Y-39983 on axonal regeneration in the crushed optic nerve in vivo. Immediately after crushing the left optic nerve, Y-39983 was injected into the vitreous and the crushed site. An injection of 10 μM Y-39983 induced the crushed axons to regenerate and pass over the crush site. In contrast, very few axons passed beyond the crush site in the optic nerve with phosphate-buffered saline injection. The second injection of 10 μM Y-39983 on day 7 doubled the number of regenerated axons, suggesting that new axons may have entered into the optic nerve after day 7 and that a continuous supply of the drug may make more axons to regenerate.

Unilateral optic nerve crush induces bilateral retinal glial cell proliferation

The post-injury responses of retinal ganglion cells elicit a number of glial reactions which have not been completely understood. The bilateral pattern of non-neuronal retinal cell proliferation was examined in association with the differential fates of unilaterally injured adult retinal ganglion cells by means of bromodeoxyuridine (BrdU) immunocytochemistry. Lateralization of the glioproliferative events was studied by analysing both the experimental and the uninjured contralateral as well as matched retinas of sham-operated animals. Control adult rat retina included very few BrdU-positive cells within the nerve fibre and ganglion cell layers; however, experimental retinas of degenerating groups exhibited statistically significantly higher densities of newborn cells in most layers. Clusters of labelled cells were found in the inner plexiform layer related to OX-42 staining, indicating their microglial nature. Indeed, double-labelling experiments, after short-term unilateral optic nerve crushing, identified proliferating retinal glial cells in vivo. Both types of glia, astroglial and microglial cells, exhibited BrdU-positive labelling in injured as well as uninjured experimental rat retinas. Moreover, microglial proliferating cells were also identified in explanted retinal pieces after 2 days in culture. Affected and contralateral retinas responded similarly to the unilateral experimental manipulations applied with respect to BrdU labelling. The acute glial responses observed suggest that bilateral glial proliferation might represent a common response related to degeneration events in both retinas, i.e. ipsi- and contralateral to the experimental injury.

Integration between Abutting Retinas: Role of Glial Structures and Associated Molecules at the Interface

Integration between subretinal grafts and the host retina is limited in part by the presence of a barrier at the graft-host interface. This study was conducted to identify factors that may contribute to this barrier, by examining the distribution of glial structures and associated molecules in different setups of overlapping retinal pieces. Neuroretinal tissue derived from mice that express green fluorescent protein (GFP) was fragmented and transplanted into the subretinal space of adult rd1 mice. In an in vitro system, two retinal pieces, derived from GFP and rd1 mice, respectively, were placed overlapping each other and forming either laminar-laminar pairs or fragment-laminar pairs. The glia-associated markers analyzed included glial fibrillary acidic protein (GFAP), cellular retinaldehyde-binding protein (CRALBP), and two molecules known to inhibit neurite outgrowth: CD44 and neurocan. Bridging fibers and migrated cells were visualized with GFP fluorescence and retinal cell markers. A thick CRALBP-immunolabeled band was observed in the interface in cultured laminar-laminar pairs, whereas a thinner band was seen in cultured fragment-laminar pairs and in transplants. Accumulation of CD44 and neurocan was also observed in the interface between abutting retinal pieces in all setups. GFP(+) bridging fibers and GFP(+) cells (some of which coexpressed neuronal markers) were observed within the abutting rd1 retina in some areas. However, such integration occurred exclusively where CRALBP, CD44, and neurocan immunolabeling appeared disrupted in the interface, but coincided with high GFAP expression within the rd1 retina. The results demonstrate that, on the one hand, an accumulation of glial-associated inhibitory molecules in the interface correlates with limited integration between overlapping retinal pieces. On the other hand, glial reactivity within the rd1 retina does not appear to be incompatible with integration.

Rabbit retinal organ culture as an in-vitro model of hepatic retinopathy.

In patients with liver insufficiency the blood ammonia concentration is elevated, which induces structural and functional retinal alterations referred to as "hepatic retinopathy". The fact that some of these alterations are reversible after timely liver transplantation rationalizes therapeutic/preventive approaches to maintain the retina before a donor organ is available. Here, we describe further elaboration and characterization of our retinal organ culture model. Retinal pieces of neonatal rabbits were explanted and cultured for up to 22 days. After 7 days in vitro (DIV7), some of the cultures were exposed to 0.25, 0.5, 1.0, 3.0, or 7.0 mM ammonia for varying periods of time and then studied by light and electron microscopy. Untreated (control) cultures underwent several changes during prolonged culturing; these included increased expression of GFAP and of Bcl-2, and decreased levels of glutamine synthetase, in Müller (glial) cells. However, the retinal layering remained intact and even some retinal ganglion cells survived although their axons had been cut. Exposure to elevated levels of ammonia caused morphologic alterations in the explants compatible with those noted in hepatic retinopathy in vivo. Specifically, we observed neuronal cellular degeneration with destruction of retinal layers and formation of rosettes, as well as decreased expression of intermediate filament proteins, migration of cell nuclei, cell swelling, and up-regulation of glutamine synthetase immunoreactivity in (some) Müller cells. Our findings indicate that the organ culture model may be a useful tool for detailed studies on the cellular and subcellular mechanisms underlying hepatic retinopathy and for in vitro testing of measures to alleviate HR symptoms.

Neuronal integration in an abutting-retinas culture system.

Limited integration is consistently observed between subretinal transplants and host retinas. In the current study, an in vitro model system for studying connections forming between two abutting retinas was developed. Neuroretinas were dissected from normal wild-type (WT) mice and green fluorescent protein (GFP) transgenic mice (obtained at postnatal days [P]0, P5, or P60), as well as from adult rd mice. Pieces from two different retinas (WT-WT, GFP-WT, GFP-rd) were placed side-by-side (contacting each other at the margins) or overlapping each other in organ cultures for 7 or 12 days. The abutting retinal pieces derived from animals of the same age (P5-P5; P60-P60) or of different ages (P0-P60; P5-P60). Retinal cells and fibers were visualized in wholemount preparations and in cross sections by immunocytochemistry using antibodies against neurofilament (NF+), neuronal nitric oxide synthase (NOS+), and protein kinase C (PKC+) and by GFP fluorescence (GFP+). In side-by-side pairs (WT-WT, GFP-WT), numerous horizontal cell fibers (NF+) and amacrine cell fibers (NOS+) crossed the interface between the two pieces, forming continuous plexiform layers. In overlapping pairs, NF+, NOS+, and PKC+ fibers displayed parallel plexiform layers, and no crossover of fibers was observed in any of the pair combinations examined (WT-WT, GFP-WT, GFP-rd). Some integration was seen only in small areas where the structure of both retinal pieces was disrupted at the interface. The results demonstrate the ability of neurites to extend between abutting retinas and to make appropriate target choices when they are placed side-by-side. However, this ability is limited when they overlap each other, similar to that observed in subretinal transplantation.

Selective staining by vital dyes of Müller glial cells in retinal wholemounts.

Müller glial cells within the retina may respond to different signaling molecules with an elevation of their intracellular free calcium. To prove the localization of the recorded calcium responses in Müller cells within acutely isolated retinal wholemounts, retinal pieces from adult animals and humans were exposed to different vital dyes just after the calcium imaging records were finished. The dyes, Mitotracker Orange, Mitotracker Green, Celltracker Orange, Celltracker Green, and monochlorobimane, are all selectively taken up by Müller glial cells, while neuronal cells remain largely devoid of the dyes. By using this method, it can be demonstrated that the free calcium alterations within the wholemounts indeed occur within Müller cells. Moreover, the cross-sectional areas of (dye-filled) Müller glial cell bodies, as well as of (dye-free) neuronal cell bodies, can be measured in retinal wholemounts, and the spatial densities of both types of cells can be determined. The vital dye loading of Müller cells may facilitate investigations of stimulus-induced alterations of retinal glial cell physiology and morphology.

Distribution of the alpha1 subunit of the GABA(A) receptor on midget and parasol ganglion cells in the retina of the common marmoset Callithrix jacchus.

The inhibitory neurotransmitter gamma aminobutyric acid (GABA) has been shown to influence the responses of ganglion cells in the mammalian retina. Consistently, GABA(A) receptor subunits have been localized to different ganglion cell types. In this study, the distribution of the alpha1 subunit of the GABA(A) receptor on the dendrites of midget and parasol ganglion cells was investigated quantitatively in the retina of a New World monkey, the marmoset. Ganglion cells were injected with Neurobiotin in a live in vitro retinal whole-mount preparation. Retinal pieces were then processed with an antibody against the alpha1 subunit of the GABA(A) receptor. Strong punctate immunoreactivity indicative of synaptic localization is present in the ON and OFF sublamina of the inner plexiform layer. Many of the immunoreactive puncta coincide with the dendrites of both midget and parasol ganglion cells. Immunoreactive puncta are present on distal and proximal dendrites of ON and OFF cells of both ganglion cell types. On average, parasol cells show a slight increase in the spatial density of immunoreactive puncta with distance from the soma, whereas the density of immunoreactive puncta on midget cells stays even. Parasol ganglion cells show a slightly higher average density of immunoreactive puncta (0.083 puncta/microm dendrite) than midget cells (0.054 puncta/microm dendrite).

Flupirtine ameliorates ischaemic-like death of rat retinal ganglion cells by preventing calcium influx

The effect of flupirtine on the loss of retinal ganglion cells following transient elevation of intraocular pressure (experimental ischaemia) or NMDA-induced excitotoxicity was studied. Ischaemia (60 min) or intravitreal injection of NMDA (20 nmol) caused a decrease in Thy-1 mRNA and Thy-1 immunoreactivity which are associated with ganglion cells. Administration of flupirtine counteracted these changes. Moreover, flupirtine dose-dependently inhibited NMDA-induced 45 Ca 2+ influx into cultured cortical neurones and retinal pieces in vitro with maximal inhibition being observed at 200 μM. A similar concentration of flupirtine failed to inhibit kainate-stimulated calcium influx into cultured cortical neurones. In addition, flupirtine had no significant effect on [ 3 H ]nitrendipine or [ 3 H ]diltiazem binding to cortical membranes. The present studies are consistent with previous findings which suggested flupirtine to act as a NMDA antagonist by a mechanism that still remains to be clarified.

Midget and parasol ganglion cells of the primate retina express the alpha1 subunit of the glycine receptor.

Glycine is a major inhibitory neurotransmitter in the mammalian retina and has been shown to influence the responses of ganglion cells. Midget and parasol ganglion cells serve distinct physiological roles in the primate retina and show differences in their response characteristics to light stimuli. In the present study, we addressed the question of whether the expression of glycine receptors differs in midget and parasol ganglion cells. Ganglion cells in the retinae of marmoset and macaque monkeys were injected with Neurobiotin in a live in vitro retinal whole-mount preparation. Retinal pieces were then processed with an antibody against the alpha1 subunit of the glycine receptor. Strong punctate immunoreactivity indicative of synaptic localization is present in the ON and OFF sublamina of the inner plexiform layer. Many of the immunoreactive puncta coincide with the dendrites of both midget and parasol ganglion cells. Immunoreactive puncta are present on distal and proximal dendrites of ON and OFF cells. These results suggest that ON and OFF midget and parasol cells do not differ with respect to the distribution of the alpha1 subunit of the glycine receptor.

Neurite growth‐inhibitory properties of amyloid β-peptides in vitro: Aβ25-35, but not Aβ1-40, is inhibitory

The effects of freshly-solubilized synthetic amyloid β-peptides on the growth of neurites from high density cultures of embryonic chick dorsal root ganglia and retinal pieces over periods of 24 to 48 hours were examined. The undecapeptide β25–35, the presumed neurotoxic moiety of β1–40, was found to inhibit neurite outgrowth, whereas β1–40 and β1–28 were without effect. The growth-inhibitory effect of β25–35 was apparent at a concentration of 10 ng/ml and showed concentration dependence. The inhibitory effect was reversible, since dorsal root ganglia grown initially in β25–35-supplemented medium showed more extensive neurite outgrowth when transferred to normal growth medium after 24 hours than dorsal root ganglia maintained in freshly-made β25–35-supplemented medium throughout. It is concluded that β25–35 has neurite growth-inhibitory properties in vitro not shared by β1–40 or β1–28. The differences in peptide behaviour may be related to aggregation state. These findings may have implications for understanding plaque morphology in normal ageing and Alzheimer's disease, since N-terminally truncated amyloid β-peptides are preferentially localized in the diffuse plaques which lack a surrounding neuritic reaction; this may also indicate a physiological neurite growth-inhibitory activity for N-terminally truncated amyloid β-peptides. © 1997 John Wiley & Sons, Ltd.

Neurite growth-inhibitory properties of amyloid β-peptidesin vitro: Aβ25-35, but not Aβ1-40, is inhibitory

The effects of freshly-solubilized synthetic amyloid β-peptides on the growth of neurites from high density cultures of embryonic chick dorsal root ganglia and retinal pieces over periods of 24 to 48 hours were examined. The undecapeptide β25–35, the presumed neurotoxic moiety of β1–40, was found to inhibit neurite outgrowth, whereas β1–40 and β1–28 were without effect. The growth-inhibitory effect of β25–35 was apparent at a concentration of 10 ng/ml and showed concentration dependence. The inhibitory effect was reversible, since dorsal root ganglia grown initially in β25–35-supplemented medium showed more extensive neurite outgrowth when transferred to normal growth medium after 24 hours than dorsal root ganglia maintained in freshly-made β25–35-supplemented medium throughout. It is concluded that β25–35 has neurite growth-inhibitory properties in vitro not shared by β1–40 or β1–28. The differences in peptide behaviour may be related to aggregation state. These findings may have implications for understanding plaque morphology in normal ageing and Alzheimer's disease, since N-terminally truncated amyloid β-peptides are preferentially localized in the diffuse plaques which lack a surrounding neuritic reaction; this may also indicate a physiological neurite growth-inhibitory activity for N-terminally truncated amyloid β-peptides. © 1997 John Wiley & Sons, Ltd.

Ganglion cells in the juvenile chick retina and their ability to regenerate axons in vitro

Ganglion cells in the chicken retina fail to regenerate their axons upon mechanical injury. In order to determine whether this failure to regenerate axons is intrinsic to the neurons or is mediated by the environment, we asked whether ganglion cells possess an ability to regrow their injured axons in the absence of their natural environment, namely in vitro. Since the retina contains morphologically different types of ganglion cells, it became desirable to investigate whether all types of ganglion cells contribute to regeneration of axons. Ganglion cells were labelled post-mortem with the fluorescent dye DiI and described morphologically. Morphometric parameters like the sizes of their perikarya, their dendrites, and the patterns of dendritic ramification and stratification were considered for grouping cells. Although a strong classification of the cells could not be achieved because of the high diversity among this population of neurons, the chick retinal ganglion cells could be separated into seven regular groups which have their somata located within the ganglion cell layer and into one group with the somata located within the inner nuclear layer (displaced ganglion cells). The experimental procedure for regeneration combines crush injury of the chick optic nerve in situ with explantation of retinal pieces 1 week later for organ cultures in a serum-free medium. Under these conditions, the ganglion cells extended axons 1 day after explantation on polylysine/laminin. The densities of ganglion cells contributing to the axonal regrowth reached up to 1447 cells mm −2 (mean 1028 cells mm −2; s.d. 237). This density correspons to 13% of the ganglion cell density in the normal retina, averaged across the total retina area. Although the dendrites of some cells whose axons had regenerated were altered in comparison with the normal ganglion cells, all morphological types of ganglion cells including those with displaced cell bodies contributed propotionally to the regrowth of axons from the explants.

Antiserum to lucifer yellow: preparation, characterization, and use for immunocytochemical localization of dye-filled retinal neurons.

We present a new method for the preparation of antisera to Lucifer Yellow, and these antisera are here shown to be particularly suitable for immunocytochemical localization of multiple dye-injected cells in large pieces of vertebrate retina. The method involves the preparation of covalent conjugates of the VS isomer of Lucifer Yellow with keyhole limpet hemocyanin (KLH) or rabbit serum albumin (RSA), and their use as immunogens in rabbits. Both carrier protein conjugates yielded robust antibody responses. Antiserum to the KLH-LY conjugate contained precipitating antibodies against LY and KLH, although activity to the latter did not interfere with immunocytochemical staining. Rabbit antiserum to the RSA-LY conjugate contained precipitating antibody only against LY. When used for immunocytochemical staining of large retinal pieces containing many LY-filled cells, both antisera yielded well-stained, darkly filled cells similar to those seen with the Golgi technique; even very fine dendritic processes of retinal ganglion cells could be followed for long distances. LY immunocytochemistry provides a useful alternative to photooxidation for the analysis of multiple dye injected cells, especially in whole mounts. This approach may also be useful for immunocytochemical identification of cells filled with LY after tissue fixation.

Mechanisms governing neuronal degeneration and axonal regeneration in the mature retinofugal system

The ability of mammalian CNS neurons to regrow their lesioned axons declines during late embryonic and postnatal development. Consequently, adult retinal ganglion cells of mammals respond to injuries with rapid anterograde and protracted retrograde (Wallerian) degeneration. To monitor the cascade of events initiated by neuronal injuries, and to explore whether the regressive events of this cascade can be blocked or reversed, axotomy-induced ganglion cell responses were investigated in adult rats. The aim of the experiments was to block degradation of axotomized ganglion cells with enzymes which inhibit proteolytic activities within the retina (protease inhibitors). To achieve this goal, a new fluorescence technique was employed to assess both the chronotopological pattern of degradation and the efficacy of the protease inhibitors and anti-inflammatory treatment in preventing cell death. Injection of protease inhibitors alone or combined with dexamethasone into the vitreous body of animals whose optic nerves were transected, protected ganglion cells from degradation and prevented endocytosis-dependent tracing of microglia. Two major functions of rescued ganglion cells proved their viability: (1) the numbers of ganglion cell axons extended from retinal stripes that were explanted 1 week after axotomy and cultured in vitro, were significantly higher when the retinal pieces originated from retinae pretreated with protease inhibitors and dexamethasone at the time of optic nerve transection than from untreated retinae; (2) the numbers of ganglion cells which regenerated axons into transplanted peripheral nerve pieces were more than doubled when the eyes were injected with protease inhibitors and dexamethasone during axotomy. The results show that blocking of the retinal proteases, which are presumably localized in microglial cells, and simultaneous treatment of the intraretinal inflammation, are key steps in understanding the intraretinal responses to axotomy and for beneficially manipulating the numbers of surviving neurons. In addition to the supporting influence of neurotrophic factors and to nonpermissive features of oligodendroglia, the microglia co-regulate whether neurons can regenerate their axons.

Regenerative and proliferative capacity of adult human retinal cells in vitro

The present work was undertaken (a) to determine whether adult human retinal neurons possess the ability to survive and regrow neurites in organ cultures and (b) to investigate the maintenance and proliferative activity of retinal glial cells in vitro. Using material from retinae obtained from human eyes postenucleation we established in vitro organ cultures of retinal pieces in a chemically defined, serum-free medium, previously developed for culturing adult rat and chick retinae. The time course of glial cell migration and of neurite extension was compared with that of adult rat and chick retinae which have been extensively investigated in our laboratory. It appeared from four explanted retinae that the human retinal cells survive for up to 4 weeks in culture and exhibit their typical morphologies. Immunohistochemical investigation of the migrating cells revealed that both astrocytes and Müller-like cells exhibit their typical morphologies in vitro. In explants obtained from a retina 30 years after very traumatic violence to the eye cup, the glial cells but not the neurons extended lengthy fibers. In the explants obtained from a retina about 2 months after traumatic optic nerve injury and subsequent ocular complications, several lengthy fibres were extended from the transplant's edge. They could be labeled with neurofilament antibodies but not with glial fibrillary acidic protein antibodies, indicating their neuronal origin. The results suggest that some injured human retinal neurons respond to exposure to growth-permissive substrates by regeneration of their neurites. Although some morphological features of the fibers suggest that they originate from ganglion cells in the retinal explants, definitive proof of this is not yet available.

Ability of adult rat ganglion cells to regrow axons in vitro can be influenced by fibroblast growth factor and gangliosides

The ability of lesioned adult retina ganglion cells (RGC) to survive and regrow axons in vitro was investigated in retina organ cultures under chemically defined conditions. Factors which are known to either affect the RGC survival like the basic fibroblast growth factor (FGF) or influence neurite outgrowth like gangliosides were assayed by recording the course of prelabeled RGC degeneration in vitro and the number and length of regrowing RGC axons from explanted retinal pieces. Administration of basic FGF significantly slowed down the decrease in the number of RITC-prelabeled RGC in the cultured retinae. In addition, in the presence of gangliosides (GM1, GD1a, GD1b, GT1b), the numbers of regrown RGC axons (Thy 1-immunostained) increased dramatically as compared to controls. The data indicate that adult neurons with an intrinsic ability to regenerate axons can respond to substances with neurotrophic or neurite-promoting activities in tissue cultures.

In vitro regeneration of adult rat ganglion cell axons from retinal explants.

The potential for regeneration of adult rat ganglion cell (RGC) axons was investigated in vitro. Explants from RITC (rhodamine-B-isothiocyanate) retrogradely labeled and in vivo axotomized retinae were placed on dishes coated with various substrates. The retinal pieces were cultivated in a serum-free medium and incubated under 50 to 80% O2-enriched and 5% CO2-containing atmosphere. Under these conditions, massive outgrowth of fibers was observed as early as 24 h after explantation and over a period of time extending up to 7 days in culture. By various criteria, two main types of processes could be characterized: (1) Short, thick processes emerged from either migrated flat cells or from cells inside the retinal explant, and (2) long and thin processes emerged from cells in the ganglion cell layer (GCL). By immunohistochemistry, the short processes and the cells from which they had emerged, appeared to be glial acidic fibrillary protein (GFAP)-positive Thy 1 and L 1-negative, suggesting their glial nature. The second type of long, thin processes appeared to be GFAP-negative, L1- and Thy 1-positive, indicating that they were neuronal, probably RGC processes. Proof that long fibers emerged from RGCs was provided by retrograde labeling of RGCs with RITC prior to explanation. Numerous RITC-filled RGCs survived in vitro. Regrowing axons retransported part of the accumulated fluorescent dye in the orthograde direction and thus unequivocally permitted their identification as RGC axons. The fact that adult RGC axons can reelongate in vitro might provide a useful bioassay for investigations on the factors that either support or inhibit regrowth of axons from CNS neurons.