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.     

Preparation and square wave electroporation of retinal explant cultures

This protocol details organotypic cultures of developing mouse, monkey and human retinas, which can be maintained for up to 2 weeks. Intact retinas are placed on polycarbonate filters floating on explant culture medium and fed every day with previously prepared retinal conditioned medium. Developing mouse retinas from E12.5 to P12 have been successfully cultured using this protocol as well as retinas from the equivalent stages of human and monkey development. Although this protocol does not require any special equipment, it provides a relatively high throughput. Retinal explant cultures lend themselves to complex pharmacological and genetic manipulations that are currently not feasible in vivo. A detailed procedure for square wave electroporation of retinal explants is also included to provide a high-throughput means to alter gene expression in the developing retina. This protocol for the preparation of retinal conditioned explant medium requires 4 d. Other steps of this protocol can be completed in 2 h.     

In vitro organotypic cultivation of adult newt and rat retinas

Adult rat and newt retinas were studied during long organotypic 3D cultivation. A high proliferation level was discovered in the region of growth by applying DNA synthesis markers and in vitro mitosis registration in newt retina. Aggregates were formed in the retina spheroid cavity because dedifferentiated cells migrated into this region. Small cell populations in nuclear layers also had dividing and migration capacity. Rosette formation has been shown in newt retina. It is a characteristic of fetal retinal development under pathological conditions. The antiGFAP antibody dye demonstrated an increase in the parent Müller cell population and generation of a small cell pool with short GFAP-extensions de novo. Recoverin expression studies detected its translocation from photoreceptor extensions to the cell bodies. Moreover, protein was presented in some cells inside the spheroid. It has been shown for the first time that cell proliferation occurred in the adult rat retinal spheroid developing in vitro; BrdU-positive cells and multiple mitoses were revealed in this fissue. However, the source of proliferation was not in the peripheral retina, and resident macrophages and glial cells located among neurons of the inner nuclear layer had the ability to divide. The antiGFAP antibody showed an increase in GFAP fibers in the rat retina as well as in the newt retina. Recoverin translocated into photoreceptor perikaryons and the outer plexiform layer in cultivated rat retina. Interestingly, some cells with probably de novo expression of recoverin were discovered in rat and newt inner retinas.     

Survival, excitability, and transfection of retinal neurons in an organotypic culture of mature zebrafish retina

Over the last 20 years, the zebrafish has become an important model organism for research on retinal function and development. Many retinal diseases do not become apparent until the later stages of life. This means that it is important to be able to analyze (gene) function in the mature retina. To meet this need, we have established an organotypic culture system of mature wild-type zebrafish retinas in order to observe changes in retinal morphology. Furthermore, cell survival during culture has been monitored by determining apoptosis in the tissue. The viability and excitability of ganglion cells have been tested at various time points in vitro by patch-clamp recordings, and retinal functionality has been assessed by measuring light-triggered potentials at the ganglion cell site. Since neurogenesis is persistent in adult zebrafish retinas, we have also monitored proliferating cells during culture by tracking their bromodeoxyuridine uptake. Reverse genetic approaches for probing the function of adult zebrafish retinas are not yet available. We have therefore established a rapid and convenient protocol for delivering plasmid DNA or oligonucleotides by electroporation to the retinal tissue in vitro. The organotypic culture of adult zebrafish retinas presented here provides a reproducible and convenient method for investigating the function of drugs and genes in the retina under well-defined conditions in vitro.     

The perplexed and confused mutations affect distinct stages during the transition from proliferating to post-mitotic cells within the zebrafish retina

To identify and study genes essential for vertebrate retinal development, we are screening zebrafish embryos for mutations that disrupt retinal histogenesis. Key steps in retinogenesis include withdrawal from mitosis by multipotent neuroepithelial cells, specification to particular cell types, migration to the appropriate laminar positions, and molecular and morphological differentiation. In this study, we have identified two recessive mutations that affect the transition of proliferating neuroepithelial cells to postmitotic retinal cells. Both the perplexed and confused mutant phenotypes were initially detectable when the first retinal neuroepithelial cells began to leave the cell cycle. At this time, each mutant retina showed increased cell death and a lack of morphological differentiation. Cell death was found to be apoptotic in both perplexed and confused retinas based on TUNEL analysis and activation of caspase-3. TUNEL-phosphoRb-BrdU colocalization studies indicated that the perplexed mutation caused death in cells transitioning from a proliferative to postmitotic state. For the confused mutation, TUNEL-phosphoRb-BrdU analysis revealed that only a subset of postmitotic cells were induced to activate apoptosis. Mosaic analysis demonstrated that within the retina the perplexed mutation functions noncell-autonomously. Furthermore, whole lens or eye cup transplantations indicated that the retinal defect was intrinsic to the retina. Mosaic analysis with confused embryos showed this mutation acts cell-autonomously. From these studies, we conclude that the perplexed and confused genes are essential at distinct stages during the transition from proliferating to postmitotic cells within the zebrafish retina.     

Optimization of Babesia bovis transfection methods

The tick borne Babesia parasites remain an important limitation for development of cattle industries worldwide. A stable transfection of Babesia bovis will be useful for functional analysis of the recently sequenced B. bovis genome and to design improved methods to control Babesia infections. In this study, we describe a novel system for nucleofection of B. bovis infected erythrocytes and we optimize methods to introduce plasmids encoding the luciferase reporter gene into Babesia infected erythrocytes or free merozoites using either a BioRad GenePulser II electroporation system or nucleofection technology (Amaxa) A comparative study among four different transfection methods: transfection of infected erythrocytes and purified merozoites with 2 or 100 microg of plasmid, using electroporation (BioRad GenePulser II) or nucleofection (Amaxa) indicates that electroporation of infected erythrocytes with 100 microg of plasmid or nucleofection with 2 microg of plasmid are the most efficient ways to transfect B. bovis parasites. The data also indicate that nucleofection is more efficient than electroporation for transfecting small quantities of plasmids (2 microg range), whereas the inverse is true for transfection of larger quantities (100 microg range). This information will facilitate further development of efficient stable transfection systems.     

Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration

The death of photoreceptor cells caused by retinal degenerative diseases often results in a complete loss of retinal responses to light. We explore the feasibility of converting inner retinal neurons to photosensitive cells as a possible strategy for imparting light sensitivity to retinas lacking rods and cones. Using delivery by an adeno-associated viral vector, here, we show that long-term expression of a microbial-type rhodopsin, channelrhodopsin-2 (ChR2), can be achieved in rodent inner retinal neurons in vivo. Furthermore, we demonstrate that expression of ChR2 in surviving inner retinal neurons of a mouse with photoreceptor degeneration can restore the ability of the retina to encode light signals and transmit the light signals to the visual cortex. Thus, expression of microbial-type channelrhodopsins, such as ChR2, in surviving inner retinal neurons is a potential strategy for the restoration of vision after rod and cone degeneration.     

Local regeneration in the retina of the goldfish

We have studied regeneration of the retina in the goldfish as a model of regenerative neurogenesis in the central nervous system. Using a transsclearal surgical approach, we excised small patches of retina that were replaced over several weeks by regeneration. Lesioned retinas from three groups of animals were studied to characterize, respectively, the qualitative changes of the retina and surrounding tissues during regeneration, the concomitant cellular proliferation, and the quantitative relationship between regenerated and intact retina. The qualitative and quantitative analyses were done on retinas prepared using standard methods for light microscopy. The planimetric density of regenerated and intact retinal neurons was computed in a group of animals in which the normal planimetric density ranged from high to low. Cell proliferation was investigated by making intraocular injections of 5-bromo-2′-deoxyuridine (BUdr) at various survival times to label proliferating cells and processing retinal sections for BUdr immunocytochemistry. The qualitative analysis showed that the surgery created a gap in the existing retina that was replaced with new retina over the subsequent weeks. The BUdr-labeling experiments demonstrated that the excised retina was replaced by regeneration of new neurons. Neuroepithiallike cells clustered on the wound margin and migrated centripetally, appositionally adding new retina to the old. The quantitative analysis showed that the planimetric density of the regenerated neurons approximated that of the intact ones.     

Local regeneration in the retina of the goldfish.

We have studied regeneration of the retina in the goldfish as a model of regenerative neurogenesis in the central nervous system. Using a transscleral surgical approach, we excised small patches of retina that were replaced over several weeks by regeneration. Lesioned retinas from three groups of animals were studied to characterize, respectively, the qualitative changes of the retina and surrounding tissues during regeneration, the concomitant cellular proliferation, and the quantitative relationship between regenerated and intact retina. The qualitative and quantitative analyses were done on retinas prepared using standard methods for light microscopy. The planimetric density of regenerated and intact retinal neurons was computed in a group of animals in which the normal planimetric density ranged from high to low. Cell proliferation was investigated by making intraocular injections of 5-bromo-2'-deoxyuridine (BUdr) at various survival times to label proliferating cells and processing retinal sections for BUdr immunocytochemistry. The qualitative analysis showed that the surgery created a gap in the existing retina that was replaced with new retina over the subsequent weeks. The BUdr-labeling experiments demonstrated that the excised retina was replaced by regeneration of new neurons. Neuroepithial-like cells clustered on the wound margin and migrated centripetally, appositionally adding new retina to the old. The quantitative analysis showed that the planimetric density of the regenerated neurons approximated that of the intact ones.     

Ultrasound-mediated gene transfer into neuronal cells

A new field of gene transfer is emerging as a simple, effective means to drive the expression foreign genes in cells: ultrasound-mediated gene transfer or sonoporation. We report here that sonoporation is an effective means of gene transfer for cultured neurons, a cell type that has been difficult to transfect. Neuronal cell types that are effectively sonoporated include chick retinal neurons, chick dorsal forebrain, chick optic tectum, PC12 cells, rat cerebellar neurons and mouse hippocampal neurons. Depending on the type of cell and conditions of sonoporation the transfection efficacy was as high as 20%. Sonoporation of plasmid DNA was effective for cells adherent to a substrate and for free-floating cells that were freshly dissociated. In the free-floating preparations, between 60 and 95% of the cells that were transfected were neuronal, as much as 90% higher than that observed for other methods of gene transfer including adenovirus and lipid-based transfection methods. We conclude that sonoporation is a simple, effective and inexpensive means by which to preferentially transfect DNA into neuronal cells.     

High-efficiency transfection of mammalian neurons via nucleofection

Transfection of foreign DNA is widely used to study gene function. However, despite the development of numerous methods, the transfer of DNA into postmitotic cells, such as neurons, remains unsatisfactory with regard to either transfection efficiency or cytotoxicity. Nucleofection overcomes these limitations. Direct electroporation of expression plasmids or oligonucleotides into the nucleus ensures both good cell viability and consistently high transfection rates. This allows biochemical analyses of transfected neurons, for example, western blot analyses of protein levels after RNA interference (RNAi) knockdown or microRNA transfection. We provide comprehensive protocols for performing nucleofection with high efficiency on primary neurons. The focus is on the recently developed 96-well shuttle system, which allows the simultaneous testing of up to 96 different plasmids or experimental conditions. Using this system, reproducible high-throughput expression of various transgenes is now feasible on primary neurons, for example large-scale RNAi analyses to downregulate gene expression. The protocol typically takes between 2 and 3 h.     

Trefoil factor family peptide 2 acts pro-proliferative and pro-apoptotic in the murine retina

Although expression of trefoil factor family (TFF) peptides has been reported in the brain, nothing is known about TFF expression in the retina. The aim of this study was to test whether TFF peptides are expressed in the murine retina and have any function here. In contrast to most tissues studied, where TFF1 and TFF3 are the predominant peptides, TFF2 is the only peptide expressed in the murine retina. Immunohistochemical studies on murine retinal sections indicate that cells of the ganglion cell layer are the retinal source for murine TFF2 (Tff2). In organotypic murine retina cell cultures recombinant TFF2 exerted a strong pro-apoptotic and pro-proliferative rather than an anti-apoptotic and anti-proliferating effect described in most human cancer cell lines investigated so far. In blockage experiments we were able to demonstrate that the pro-apoptotic effect of TFF2 is caspase-dependent. Western blot analysis of TFF2 treated retinal wholemount homogenates revealed significant reductions in the phosphorylation level of ERK and STAT3 proteins compared to basal conditions, suggesting that in the developing murine retina survival mechanism are down-regulated upon TFF2 administration. Our results suggest that during retinal cell death periods, requiring a tightly regulated balance between cell survival and cell death, TFF2 acts pro-proliferative and pro-apoptotic at least in developing mouse retinae cultured in vivo.     

Organotypic culture of neural retina as a research model of neurodegeneration of ganglion cells

Organotypic models deserve special attention among the large variety of methods of vertebrate retina cultivation. The purpose of this study was to make a detailed qualitative and quantitative characterization of a model employing roller organotypic cultivation of the neural retina of rat eye posterior segment, with special attention to morphological and functional characteristics of retinal ganglion cells. The study included morphological analysis of retina histological preparations as well as estimation of RNA synthesis and evaluation of neuron survival by the Brachet and TUNEL methods, respectively. Retina has been shown to display normal morphofunctional characteristics for the first 12 h of cultivation. After 24 h, a substantial number of ganglion cells underwent pyknosis and stopped RNA synthesis. Almost all the cells of the retinal ganglion layer became apoptotic by 3–4 days in vitro. In the course of cultivation, neural retina is detached from the underlying layers of the posterior eye segment and undergoes significant cytoarchitectonic changes. The causes of ganglion cell death during organotypic cultivation of eye posterior segment are discussed. This method can serve as a suitable model for the screening of new retinoprotectors and for research on ganglion cell death resulting from retina degenerative diseases, e.g. glaucoma.     

Cellular determination in the Xenopus retina is independent of lineage and birth date

Xenopus embryos injected with tritiated thymidine throughout the stages of embryonic retinal neurogenesis showed that more than 95% of the embryonic retinal cells are born within a 25 hr period. While there are shallow central to peripheral, dorsal to ventral, and interlaminar gradients of neurogenesis in these eyes, throughout most of this 25 hr period, postmitotic cells are being added to all sectors and layers. Small clones of differentiated retinal neurons and glia derived from single neuroepithelial cells injected with HRP. These clones were elongated radially. They were also composed of many different combinations of cell types, suggesting a mechanism whereby determination is arbitrarily and independently assigned to postmitotic cells. Such a model, when tested statistically, fits our data very well. We present a scheme for cellular determination in the Xenopus retina in which a coherent group of clonally related cells stretch out radially as lamination begins. This brings different cells into different microenvironments. Local interactions in these microenvironments then lead the cells toward specific fates.     

Type-1, but Not Type-5, Metabotropic Glutamate Receptors are Coupled to Polyphosphoinositide Hydrolysis in the Retina

mGlu1 and mGlu5 metabotropic glutamate receptors are expressed in the vertebrate retina, and are co-localized in some retinal neurons. It is believed that both receptors are coupled to polyphosphoinositide (PI) hydrolysis in the retina and their function may diverge in some cells because of a differential engagement of downstream signaling molecules. Here, we show that it is only the mGlu1 receptor that is coupled to PI hydrolysis in the retina. We used either bovine retinal slices or intact mouse retinas challenged with the mixed mGlu1/5 receptor agonist, DHPG. In both models, DHPG-stimulated PI hydrolysis was abrogated by the selective mGlu1 receptor antagonist, JNJ16259685, but was insensitive to the mGlu5 receptor antagonist, MPEP. In addition, the PI response to DHPG was unchanged in the retina of mGlu5^?/? mice but was abolished in the retina of crv4 mice lacking mGlu1 receptors. Stimulation of the mitogen-activated protein kinase pathway by DHPG in intact mouse retinas were also entirely mediated by mGlu1 receptors. Our data provide the first example of a tissue in which a biochemically detectable PI response is mediated by mGlu1, but not mGlu5, receptors. Hence, bovine retinal slices might be used as a model for the functional screening of mGlu1 receptor ligands. In addition, the mGlu1 receptor caters the potential as a drug target in the experimental treatment of degenerative disorders of the retina.

     

Creatine transporter localization in developing and adult retina: importance of creatine to retinal function

Creatine and phosphocreatine are required to maintain ATP needed for normal retinal function and development. The aim of the present study was to determine the distribution of the creatine transporter (CRT) to gain insight to how creatine is transported into the retina. An affinity-purified antibody raised against the CRT was applied to adult vertebrate retinas and to mouse retina during development. Confocal microscopy was used to identify the localization pattern as well as co-localization patterns with a range of retinal neurochemical markers. Strong labeling of the CRT was seen in the photoreceptor inner segments in all species studied and labeling of a variety of inner neuronal cells (amacrine, bipolar, and ganglion cells), the retinal nerve fibers and sites of creatine transport into the retina (retinal pigment epithelium, inner retinal blood vessels, and perivascular astrocytes). The CRT was not expressed in Müller cells of any of the species studied. The lack of labeling of Müller cells suggests that neurons are independent of this glial cell in accumulating creatine. During mouse retinal development, expression of the CRT progressively increased throughout the retina until approximately postnatal day 10, with a subsequent decrease. Comparison of the distribution patterns of the CRT in vascular and avascular vertebrate retinas and studies of the mouse retina during development indicate that creatine and phosphocreatine are important for ATP homeostasis. photoreceptor; development; glutamine synthetase; neurochemistry     

Efficient gene transfer into murine embryonic stem cells by nucleofection

Genetic manipulation of embryonic stem (ES) cells is performed by non-viral as well as viral transfection methods. We tested the recently developed nucleofection method delivering plasmid DNA directly into the nucleus for the introduction of a plasmid encoding enhanced green fluorescent protein (EGFP) into murine ES cells. Cell viability decreased from 77% before to 40% 24 h after nucleofection. Transfection effciencies in viable stem cells were between 85% and 96% with high levels of EGFP expression [mean fluorescence intensity (MFI): 630 +/- 90] 24 h after nucleofection. After a two week culture in geneticin (G418) selection medium, nearly 50% of the stem cells were EGFP positive and continued transgene expression (MFIs: 120-240) for a two further weeks. We conclude that nucleofection is an efficient nonviral gene transfer method for the introduction of genes into murine ES cells.     

Smn deficiency causes neuritogenesis and neurogenesis defects in the retinal neurons of a mouse model of spinal muscular atrophy

The eye is an excellent model for the study of neuronal development and pathogenesis of central nervous system disorders because of its relative ease of accessibility and the well‐characterized cellular makeup. We have used this model to study spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease caused by deletions or mutations in the survival of motor neuron 1 gene (SMN1). We have investigated the expression pattern of mouse Smn mRNA and protein in the neural retina and the optic nerve of wild type mice. Smn protein is present in retinal ganglion cells and amacrine cells within the neural retina as well as in glial cells in the optic nerve. Histopathological analysis in phenotype stage SMA mice revealed that Smn deficiency is associated with a reduction in ganglion cell axon and glial cell number in the optic nerve, as well as compromised cellular processes and altered organization of neurofilaments in the neural retina. Whole mount preparation and retinal neuron primary culture provided further evidence of abnormal synaptogenesis and neurofilament accumulation in the neurites of Smn‐deficient retinal neurons. A subset of amacrine cells is absent, in a cell‐autonomous fashion, in the retina of SMA mice. Finally, the retinas of SMA mice have altered electroretinograms. Altogether, our study has demonstrated defects in axodendritic outgrowth and cellular composition in Smn‐depleted retinal neurons, indicating a role for Smn in neuritogenesis and neurogenesis, and providing us with an insight into pathogenesis of SMA. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 71: 153‐169, 2011     

A mitochondria-localized glutamic acid-rich protein (MGARP/OSAP) is highly expressed in retina that exhibits a large area of intrinsic disorder

Study of retina specific genes would offer insights into retinal diseases and treatment. Based on the information from the gene expression profiles of mouse retinas, we here identified a mitochondria-localized glutamic acid-rich protein (MGARP/OSAP) as one of the highly expressed proteins in retina. Sequence analysis revealed that mouse and rat MGARPs have an extra insertion of four consecutive amino acid repeats at the C-terminus, while other homologues do not. MGARP was demonstrated to be localized to the mitochondria and overexpression of MGARP missing N-terminal region causes severe mitochondrial aggregation, implying an important role of MGARP in maintaining mitochondrial morphology. MGARP is highly expressed in mitochondria-rich layers, including inner segment of the photoreceptor, outer plexiform layer and ganglion cell layers of mouse retina. Far-UV CD spectrum analysis suggested that MGARP exhibits a large area of intrinsic disorder and the unusual position of its Tyr fluorescence suggested that Tyr residues in MGARP might form excimer and exist in an ionized state. These findings implied that MGARP be a good candidate for assembling certain ion channels on mitochondria membrane and have great potential to be involved in retinal energetic metabolism through mitochondria related pathway.     

Prion protein expression differences in microglia and astroglia influence scrapie-induced neurodegeneration in the retina and brain of transgenic mice

Activated microglia and astroglia are known to be involved in a variety of neurodegenerative diseases, including prion diseases. In the present experiments, we studied activation of astroglia and microglia after intraocular scrapie infection in transgenic mice expressing prion protein (PrP) in multiple cell types (tg7 mice) or in neurons only (tgNSE mice). In this model, scrapie infection and protease-resistant PrP deposition occurs in the retinas of both strains of mice, but retinal degeneration is observed only in tg7 mice. Our results showed that the retinas of tg7 and tgNSE mice both had astroglial activation with increased chemokine expression during the course of infection. However, only tg7 retinas exhibited strong microglial activation compared to tgNSE retinas, which showed little microglial activation by biochemical or morphological criteria. Therefore, microglial PrP expression might be required for scrapie-induced retinal microglial activation and damage. Furthermore, microglial activation preceded retinal neurodegeneration in tg7 mice, suggesting that activated microglia might contribute to the degenerative process, rather than being a response to the damage. Surprisingly, brain differed from retina in that an altered profile of microglial activation markers was upregulated, and the profiles in the two mouse strains were indistinguishable. Microglial activation in the brain was associated with severe brain vacuolation and neurodegeneration, leading to death. Thus, retinal and brain microglia appeared to differ in their requirements for activation, suggesting that different activation pathways occur in the two tissues.