Growth preferences of adult rat retinal ganglion cell axons in retinotectal cocultures

We examined whether regenerating axons from adult rat ganglion cells are able to recognize their appropriate target region in vitro. Explants from adult rat retina were cocultured with embryonic sagittal midbrain slices in Matrigel®. The midbrain sections contained the superior colliculus, the main target for retinal ganglion cell axons in rats, and the inferior colliculus. We observed a statistically significant preference of both temporal and nasal retinal axons to grow toward their appropriate target region (anterior and posterior superior colliculus, respectively). No preferential growth of retinal ganglion cell axons was detected in controls, for which retinal explants were cultured on their own. When retinal ganglion cell axons were given a choice between superior colliculus and inferior colliculus, axons from nasal retina preferentially grew toward the posterior superior colliculus and avoided the inferior colliculus. In contrast, temporal axons in the same assay did not show preference for either of the colliculi. These findings suggest that regenerating axons from adult rat retina are able to recognize target-specific guidance cues released from embryonic midbrain targets in vitro. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 379–387, 1998     

Target‐derived and locally derived neurotrophins support retinal ganglion cell survival in the neonatal rat retina

Brain‐derived neurotrophic factor (BDNF) and neurotrophin‐4/5 (NT‐4/5) protein and mRNA are found in the neonatal rat retina and also in target sites such as the superficial layers of the superior colliculus. Both neurotrophins support neonatal retinal ganglion cell survival in vitro. In vivo, injections of recombinant BDNF and NT‐4/5 reduce naturally occurring cell death as well as death induced by removal of the contralateral superior colliculus. In the latter case, the peak of retinal ganglion cell death occurs about 24 h postlesion. We wished to determine: whether a similar time‐course of degeneration occurs after selective removal of target cells or depletion of target‐derived trophic factors, and whether ganglion cell viability also depends on intraretinally derived neurotrophins. Retinal ganglion cell death was measured 24 and 48 h following injections of kainic acid or a mixture of BDNF and NT‐4/5 blocking antibodies into the superior colliculus and 24 h after intraocular injection of the same antibodies. Retinotectally projecting ganglion cells were identified by retrograde labeling with the nucleophilic dye diamidino yellow. We show that collicular injections of either kainic acid or BDNF and NT‐4/5 blocking antibodies significantly increased retinal ganglion cell death in the neonatal rat 24 h postinjection, death rates returning to normal by 48 h. This increase in death was greatest following collicular injections; however, death was also significantly increased 24 h following intravitreal antibody injection. Thus retinal ganglion cell survival during postnatal development is not only dependent upon trophic factors produced by central targets but may also be influenced by local intraretinal neurotrophin release. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 319–327, 2004     

Induction of α1-tubulin gene expression during development and regeneration of the fish central nervous system

The α1- and α2-tubulin encoding genes were cloned from a goldfish genomic DNA library. α1- and α2-tubulin RNA expression was examined in developing and adult retinas. These studies demonstrated increased α1-tubulin RNA in presumptive ganglion cells that grow axons early in retinal development and in adult retinal ganglion cells whose optic axons had been damaged. The α2-tubulin RNA was undetectable in developing retina and constitutively expressed in adult retinal ganglion cells regardless of optic nerve crush. To determine if these changes in α1-tubulin RNA reflected changes in α1-tubulin promoter activity, we introduced into zebrafish embryos and adult goldfish retinal explants expression vectors harboring the α1-tubulin gene's promoter. These studies showed that the α1-tubulin promoter confers a developmentally regulated, neuron-restricted pattern of reporter gene expression in vivo and its activity is increased in adult retinal neurons induced to regenerate their axons. Promoter deletions defined regions of α1-tubulin DNA necessary for this pattern of expression. These results suggest that DNA sequences necessary for α1-tubulin gene induction during central nervous system development and regeneration are contained within the α1-tubulin gene's 5′-flanking DNA and that this promoter will be useful for identifying these elements and their DNA binding proteins. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 429–440, 1998     

Regeneration of functional synaptic connections between widely separated neurons in the adult mammalian central nervous system

The responses to light of retinal ganglion cells with regenerated axons can be recorded from axons teased from peripheral nerve grafts replacing the optic nerve of the adult rat or hamster. These responses resemble those of normal retinal ganglion cells but can no longer be observed several months after grafting, concomitant with ongoing loss of the population of axotomized retinal ganglion cells. Synapses formed with neurons in the superior colliculus by retinal ganglion cell axons regenerated through peripheral nerve grafts mediate both excitatory and inhibitory responses. These experiments demonstrate that when provided with an appropriate milieu for elongation, neurons indigenous to the adult mammalian central nervous system can make functional reconnections with distant targets within the nervous system.     

Cytochrome <Emphasis Type="Italic">c</Emphasis> Release and Caspase-3 Activation in Retinal Ganglion Cells Following Different Distance of Axotomy of the Optic Nerve in Adult Hamsters

This study examined the relationship between the distance of axotomy and the death of injured retinal ganglion cells (RGCs) in adult hamsters and the relationship of cytochrome c and caspase-3 on the death pathway of RGCs. The left optic nerve (ON) of adult hamsters was transected either at 1 or 3 mm away from the optic disc, and retrogradely labeled with Flurogold on the ON stump. After a predetermined period of postoperative time, the surviving RGCs were counted by retina flat-mount, and the activation of cytochrome c and caspase-3 were investigated by immunohistochemistry. Cell loss was found to be much faster (P < 0.01), more cells with cytochrome c were observed (P < 0.05) and the activation of caspase-3 was earlier when ON was transected 1 mm away from the optic disc than when was transected 3 mm away from the optic disc. Distance of axotomy affects the axotomized cell death rate where more RGCs died when the ON transection was applied closer to the eye. The timing of activation of caspase-3 in the RGCs may be linked to the distance of axotomy.Special issue dedicated to Dr. Lawrence F. Eng     

Cellular interactions determine neuronal phenotypes in rodent retinal cultures

Progenitor cells isolated from early rat embryo retinas differentiate into phenotypes normally generated early in retinal development (e.g., ganglion cells), whereas progenitors isolated from postnatal retinas differentiate into later-generated retinal cell types (e.g., rod photoreceptors; Reh and Kljavin, J. Neurosci. 9:4179–4189; 1989; Adler and Hatlee, 1989; Science 243:391–393; Sparrow, Hicks, and Barnstable, 1990, Dev. Brain Res. 51:69–84). To determine whether this change in committment is intrinsic to the progenitor cells, or alternatively can be modified by interactions with their developing environment, I co-cultured mouse and rat retinal cells, from different developmental stages, and identified the resulting phenotypes with species-specific and cell class-specific antibodies. I found that the phenotypes into which mouse neuroepithelial cells differentiate depends on the phenotypes of the rat cells that surround them. Retinal precursor cells from embryonic day (E) 10–12 will adopt the rod photoreceptor phenotype only when close to cells expressing this phenotype. By contrast, when the E10–12 retinal progenitor cells are cultured with cells from the cerebral cortex, they differentiate primarily into large multipolar neurons, similar in their morphology and antigen expression to retinal ganglion cells. These results indicate that interactions among the cells of the developing retina are important in the determination of cell fate. © 1992 John Wiley & Sons, Inc.     

Agmatine protects cultured retinal ganglion cells from tumor necrosis factor-alpha-induced apoptosis

AimsWe investigated the protective effects of agmatine against tumor necrosis factor (TNF)-α-induced apoptosis in transformed rat retinal ganglion cells (RGC-5 cell line).Main methodsThe RGC-5 cells were exposed to 50 ng/mL TNF-α for 48 h with or without presence of 100 μM agmatine as indicated. Cell viability was determined by lactate dehydrogenase (LDH) assay. Double staining with Hoechst 33342 and propidium iodide for morphological analysis was performed. Subsequently, using annexin V assay, the proportion of cells actively undergoing apoptosis was determined.Key findingsAfter 48 h of exposure to 50 ng/mL TNF-α, 17.00% of RGC-5 cells were lost, as evident by LDH assay. TNF-α-induced RGC-5 cell death was reduced to 8.14% with 100 μM agmatine treatment. This observed cell loss was due to apoptotic cell death, as established by annexin V assay.SignificanceOur results reveal that agmatine has neuroprotective effects against TNF-α-induced apoptosis in retinal ganglion cells in vitro.     

Activation of caspase-3 in axotomized rat retinal ganglion cells in vivo.

Recently, we have shown that inhibition of caspase-3-like caspases is the most effective treatment strategy to protect adult rat retinal ganglion cells from secondary death following optic nerve transection. In the present study, we localized active caspase-3 in axotomized retinal ganglion cells in vivo and demonstrated a co-localization of the active p20 fragment and TUNEL-staining in some of these cells. In line with this, we detected an enhanced cleavage and activity of caspase-3 protein in retinal tissue after lesion, while caspase-3 mRNA expression remained unchanged. These data suggest caspase-3 as an important mediator of secondary retinal ganglion cell death following axotomy in vivo.     

Cues intrinsic to the retina induce nAchR gene expression during development

Recent studies of optic nerve regeneration in goldfish have indicated that the optic tectum plays an important role in modulating the induction of nicotinic acetylcholine receptor (nAChR) gene expression in regenerating retinal ganglion cells (Hieber, Agranoff, and Goldman, 1992, J. Neurochem. 58:1009–1015). These observations suggest that induction of these genes is regulated by brain target regions. The appearance of nAChR mRNA in the developing rat retina coincides with a time when ganglion cells are sending axons to their brain targets (Hoover and Goldman, 1992, Exp. Eye Res. 54:561–571). Might a mechanism similar to that seen during goldfish optic nerve regenerationalso mediate induction of nAChR gene expression during development of the mammalian retina? This possibility was tested by either transplanting embryonic rat retina to different brain regions, or explanting it to organ culture and assaying for nAChR gene expression. These studies showed that induction of the nAChR genes in developing rat retina is independent of the environment in which the retina develops. These results indicate that either the retinal microenvironment or a signal intrinsic to the retinal ganglion cell is responsible for this induction. © 1993 John Wiley & Sons, Inc.     

Atoh7 promotes the differentiation of Müller cells-derived retinal stem cells into retinal ganglion cells in a rat model of glaucoma

Glaucoma is one of the leading eye diseases resulting in blindness due to the death of retinal ganglion cells. This study aimed to develop novel protocol to promote the differentiation of retinal Müller cells into ganglion cells in vivo in a rat model of glaucoma. The stem cells dedifferentiated from rat retinal Müller cells were randomized to receive transfection with empty lentivirus PGC-FU-GFP or lentivirus PGC-FU-Atoh7-GFP, or no transfection. The stem cells were induced further to differentiate. Ocular hypertension was induced using laser photocoagulation. The eyes were injected with Atoh7 expression vector lentivirus PGC-FU-Atoh7-GFP. Eyeball frozen sections, immunohistochemistry, RT-PCR, Western bolt, and apoptosis assay were performed. We found that the proportion of ganglion cells differentiated from Atoh7-tranfected stem cells was significantly higher than that of the other two groups. The mean intraocular pressure of glaucomatous eyes was elevated significantly compared with those of contralateral eyes. Some retinal Müller cells in the inner nuclear layer entered the mitotic cell cycle in rat chronic ocular hypertension glaucoma model. Atoh7 contributes to the differentiation of retinal Müller cells into retinal ganglion cells in rat model of glaucoma. In conclusion, Atoh7 promotes the differentiation of Müller cells-derived retinal stem cells into retinal ganglion cells in a rat model of glaucoma, thus opening up a new avenue for gene therapy and optic nerve regeneration in glaucoma.     

Antibody that neutralizes myelin-associated inhibitors of axon growth does not interfere with recognition of target-specific guidance information by rat retinal axons

During development, many CNS projection neurons establish topographically ordered maps in their target regions. Myelin-associated inhibitors of neurite growth contribute to the confinement of fiber tracts during development and limit plastic changes after CNS projections have been formed. Neutralization of myelin-associated growth inhibitors leads to an expansion of the retinal innervation of the superior colliculus (SC). In the lesioned adult mammalian CNS, these long projection neurons are usually unable to regrow axons over long distances after lesion due to myelin-associated inhibitors, which interfere with axonal growth in vivo and in vitro. Application of a specific antibody directed against myelin-inhibitors (IN-1) promotes regrowth of corticospinal tract or retinal ganglion cell axons. In the present study, we asked whether application of an antibody to myelin-associated growth inhibitors would lead to disturbances of target-specific axon guidance. To examine this issue, we used an in vitro model, the “stripe assay,” to examine the behavior of rat retinal ganglion cell axons on membranes from embryonic and deafferented adult rat SC. On membrane preparations from embryonic rat SC, retinal fibers avoid posterior tectal membranes, possibly due to the presence of a repulsive factor. Nasal retinal axons show a random growth pattern. On membranes prepared from the deafferented adult rat SC, temporal and nasal axons prefer to grow on membranes prepared from their specific target region, which suggests the involvement of target-derived attractive guidance components. The results of the present study show that retinal axons grow significantly faster in the presence of IN-1 antibody that neutralizes myelin-associated growth inhibitors present in the membrane preparations from the adult rat SC. IN-1 antibody, however, does not interfere with specific axonal guidance. This suggests that axonal guidance and specific target finding are independently regulated in retinal axons. © 1996 John Wiley & Sons, Inc.     

T3-hydrocortisone synergism on adult-type erythroblast proliferation and T3-mediated apoptosis of larval-type erythroblasts during erythropoietic conversion in Xenopus laevis

 The conversion of an erythropoietic system from larval to adult type in anuran amphibia may possibly come about through cell replacement. The hormonal regulation of apoptosis of larval-type precursor cells and adult-type cell proliferation has yet to be examined in detail. In amphibians, corticoids synergize T₃ action during metamorphosis. In the present study, examination was made of the process of larval-to-adult conversion in the liver erythropoietic site of Xenopus laevis, with special attention to how these metamorphic hormones, T₃ and corticoid, regulate programmed cell death specific for larval erythroblasts and the proliferation of adult cells. Immunohistochemical analysis of liver sections indicates that the number of larval erythroblasts decreased to less than 50% at the early climax stage (stages 59–60) of metamorphosis. Overall liver morphology greatly changed subsequent to the climax stage from the three-lobe to the two-lobe shape. The addition of T₃ (10^-8 M) to premetamorphic tadpoles induced considerable liver morphological change and a 50% decrease in larval-type erythroblasts. These erythroblast decreases seem to take place through the apoptotic process, since double-staining experiments with in situ DNA nick-end labeling (TUNEL) and hemoglobin immunostaining revealed that DNA breakage of nuclei, a well-known feature of apoptosis, occured specifically in larval erythroblasts during prometamorphosis. Hydrocortisone (HC), which modulates T₃ action during metamorphosis, was found not to be a factor in larval cell decrease. But adult erythroblasts increased by 8 times as much through the action of T₃ and 32 times as much by the action of T₃ plus HC, indicating the important action of T₃–HC synergism. It thus follows that the erythropoietic system is converted during metamorphosis effectively by two distinct hormonal mechanisms, T₃–HC synergism on adult erythroblast proliferation and T₃-mediated programmed death of larval precursor cells. Accepted: 14 January 1999     

Maternal enrichment during pregnancy accelerates retinal development of the fetus

The influence of maternal environment on fetal development is largely unexplored, the available evidence concerns only the deleterious effects elicited by prenatal stress. Here we investigated the influence of prenatal enrichment on the early development of the visual system in the fetus. We studied the anatomical development of the rat retina, by analyzing the migration of neural progenitors and the process of retinal ganglion cell death, which exerts a key role in sculpturing the developing retinal system at perinatal ages. The number of apoptotic cells in the retinal ganglion cell layer was analyzed using two distinct methods: the presence of pyknotic nuclei stained for cresyl violet and the appearance of DNA fragmentation (Tunel method). We report that environmental enrichment of the mother during pregnancy affects the structural maturation of the retina, accelerating the migration of neural progenitors and the dynamics of natural cell death. These effects seem to be under the control of insulin-like growth factor-I: its levels, higher in enriched pregnant rats and in their milk, are increased also in their offspring, its neutralization abolishes the action of maternal enrichment on retinal development and chronic insulin-like growth factor-I injection to standard-reared females mimics the effects of enrichment in the fetuses. Thus, the development of the visual system is sensitive to environmental stimulation during prenatal life. These findings could have a bearing in orienting clinical research in the field of prenatal therapy.     

DNA replication in binucleate cells of the Malpighian tubules of Hemipteran insects

The cells of Malpighian tubules of the Hemipteran blood-sucking insect, Rhodnius prolixus, are binucleate. The cells grow without division and, at each larval moult, the DNA content of the nuclei doubles. At the final moult to the adult, however, the DNA content does not change, even though the tubules grow considerably thereafter. In contrast, the DNA content of the tubule nuclei of two other Heteropteran Hemipteran insects, Dysdercus and Oncopeltus, doubles at every moult, including the final one to the adult. If extra larval moults are induced in Rhodnius, by treatment with juvenile hormone, DNA doubling is induced at each such supernumerary larval moult. Shortly after the DNA content increases in Rhodnius tubules, the chromosomes can be seen in a condensed state; presumably, therefore, DNA replication is achieved by endomitosis. Both before this DNA doubling, and within a day after, multiple nucleoli are prominent and appear actively engaged in producing ribosomal precursors. In fed fourth stage Rhodnius, the DNA content of the tubule cell nuclei increases 5–6 days after the blood meal. Neither the rate of fluid secretion that can be induced by stimulation nor the rate of transport of p-aminohippuric acid show any change at the time of DNA replication nor in the remaining days before ecdysis to the fifth stage. Malpighian tubule cell growth without division is thus well adapted to providing, without interruption, for the excretory needs of the growing insect.     

Retinal ganglion cell topography and visual acuity of the sleepy lizard (<Emphasis Type="Italic">Tiliqua rugosa</Emphasis>)

The spatial distribution of retinal ganglion cells provides valuable insight into the importance species place on observing objects in specific regions of their visual field with higher spatial resolving power. We estimate the total number, distribution and peak density of ganglion cells in retinal wholemounts of the sleepy lizard, Tiliqua rugosa, a scincid lizard endemic to southern Australia. Ganglion cells were readily discernable from amacrine cells by their size and shape, prominent nuclei and the accumulation of Nissl-positive substances in their cytoplasm. A total of 1,654,200 (±59,400) presumed ganglion cells were estimated throughout the retina, distributed irregularly and forming a loose horizontal streak of high cell density peaking at 15,500 cells per mm². With a post nodal distance of 6.25 mm, we calculate an upper limit of visual acuity of 6.8 c/deg.     

Retinal Ganglion Cell Dendritic Atrophy in DBA/2J Glaucoma

Glaucoma is a complex disease affecting an estimated 70 million people worldwide, characterised by the progressive degeneration of retinal ganglion cells and accompanying visual field loss. The common site of damage to retinal ganglion cells is thought to be at the optic nerve head, however evidence from other optic neuropathies and neurodegenerative disorders suggests that dendritic structures undergo a prolonged period of atrophy that may accompany or even precede soma loss and neuronal cell death. Using the DBA/2J mouse model of glaucoma this investigation aims to elucidate the impact of increasing intraocular pressure on retinal ganglion cell dendrites using DBA/2J mice that express YFP throughout the retinal ganglion cells driven by Thy1 (DBA/2J.Thy1(YFP)) and DiOlistically labelled retinal ganglion cells in DBA/2J mice. Here we show retinal ganglion cell dendritic degeneration in DiOlistically labelled DBA/2J retinal ganglion cells but not in the DBA/2J.Thy1(YFP) retinal ganglion cells suggesting that a potential downregulation of Thy1 allows only ‘healthy’ retinal ganglion cells to express YFP. These data may highlight alternative pathways to retinal ganglion cell loss in DBA/2J glaucoma.     

Activation of BMP-Smad1/5/8 signaling promotes survival of retinal ganglion cells after damage in vivo

While the essential role of bone morphogenetic protein (BMP) signaling in nervous system development is well established, its function in the adult CNS is poorly understood. We investigated the role of BMP signaling in the adult mouse retina following damage in vivo. Intravitreal injection of N-methyl-D-aspartic acid (NMDA) induced extensive retinal ganglion cell death by 2 days. During this period, BMP2, -4 and -7 were upregulated, leading to phosphorylation of the downstream effector, Smad1/5/8 in the inner retina, including in retinal ganglion cells. Expression of Inhibitor of differentiation 1 (Id1; a known BMP-Smad1/5/8 target) was also upregulated in the retina. This activation of BMP-Smad1/5/8 signaling was also observed following light damage, suggesting that it is a general response to retinal injuries. Co-injection of BMP inhibitors with NMDA effectively blocked the damage-induced BMP-Smad1/5/8 activation and led to further cell death of retinal ganglion cells, when compared with NMDA injection alone. Moreover, treatment of the retina with exogenous BMP4 along with NMDA damage led to a significant rescue of retinal ganglion cells. These data demonstrate that BMP-Smad1/5/8 signaling is neuroprotective for retinal ganglion cells after damage, and suggest that stimulation of this pathway can serve as a potential target for neuroprotective therapies in retinal ganglion cell diseases, such as glaucoma.     

Antibodies against pax6 immunostain amacrine and ganglion cells and neuronal progenitors,but not rod precursors,in the normal and regenerating retina of the goldfish

Pax6 is a developmental regulatory gene that plays a key role in the development of the embryonic brain, eye, and retina. This gene is also expressed in discrete groups of neurons within the adult brain. In this study, antibodies raised against a fusion protein from a zebra fish pax6 cDNA were used to investigate the expression of the pax6 gene in the mature, growing, and regenerating retina of the goldfish. On western blots of retinal proteins, the pax6 antibodies recognize a single band at the approximate size of the zebra fish pax6 protein. In retinal sections, the antibodies label the nuclei of mature amacrine and some ganglion cells. At the retinal margin, where neurogenesis and cellular differentiation continually occur in goldfish, the antibodies label neuronal progenitors and the newly postmitotic neurons. Following injury and during neuronal regeneration, the antibodies label mitotically active progenitors of regenerating neurons. Rod precursors, proliferating cells that normally give rise solely to rod photoreceptors and are the presumed antecedents of the injury-stimulated neuronal progenitors, are not immunostained by antibodies to the pax6 protein. The results of this study document the identity of pax6-expressing cells in the mature retina and demonstrate that in the goldfish pax6 is expressed in neuronal progenitors during both retinal growth and regeneration. © 1996 John Wiley & Sons, Inc.     

Organotypic tissue culture of adult rodent retina followed by particle-mediated acute gene transfer in vitro

Background

Organotypic tissue culture of adult rodent retina with an acute gene transfer that enables the efficient introduction of variable transgenes would greatly facilitate studies into retinas of adult rodents as animal models. However, it has been a difficult challenge to culture adult rodent retina. The purpose of this present study was to develop organotypic tissue culture of adult rodent retina followed by particle-mediated acute gene transfer in vitro.

Methodology/Principal Findings

We established an interphase organotypic tissue culture for adult rat retinas (>P35 of age) which was optimized from that used for adult rabbit retinas. We implemented three optimizations: a greater volume of Ames'' medium (>26 mL) per retina, a higher speed (constant 55 rpm) of agitation by rotary shaker, and a greater concentration (10%) of horse serum in the medium. We also successfully applied this method to adult mouse retina (>P35 of age). The organotypic tissue culture allowed us to keep adult rodent retina morphologically and structurally intact for at least 4 days. However, mouse retinas showed less viability after 4-day culture. Electrophysiologically, ganglion cells in cultured rat retina were able to generate action potentials, but exhibited less reliable light responses. After transfection of EGFP plasmids by particle-mediated acute gene transfer, we observed EGFP-expressing retinal ganglion cells as early as 1 day of culture. We also introduced polarized-targeting fusion proteins such as PSD95-GFP and melanopsin-EYFP (hOPN4-EYFP) into rat retinal ganglion cells. These fusion proteins were successfully transferred into appropriate locations on individual retinal neurons.

Conclusions/Significance

This organotypic culture method is largely applicable to rat retinas, but it can be also applied to mouse retinas with a caveat regarding cell viability. This method is quite flexible for use in acute gene transfection in adult rodent retina, replacing molecular biological bioassays that used to be conducted in isolated cultured cells.