Retinal Explant Cultures Research Articles

Preparation of retinal explant cultures to study ex vivo tip endothelial cell responses

This protocol details a culture technique for neonatal mouse retina that allows the assessment and quantification of acute responses of developing blood vessels to pharmacological manipulation. The technique has proven to be a useful tool for elucidating the molecular mechanisms that underlie the guidance of tip cells in the complex scenario of the angiogenic sprouting process. This culture setting allows the acute stimulation or inhibition of cellular functions of endothelial cells in their physiological environment ex vivo. Compared with other existing techniques, such as retinal injections in animals, the explant culture described here is an easily manageable and highly flexible alternative that allows pharmacological manipulations of the developing retina vessels. The technique involves swift extraction of retina from intact eye and retinal flat mounting on a hydrophilic membrane with minimum disturbance of the tissue. The responses of tip endothelial cell sprouting activity and filopodial extension to different angiogenic and angioinhibitory factors can be evaluated within only 4 h. The whole process for the retinal explant cultures and stimulation can be completed in 10 h.

Meteorin promotes the formation of GFAP-positive glia via activation of the Jak-STAT3 pathway

Meteorin is an orphan ligand which has been previously reported to control neuritogenesis and angiogenesis, as well as gliogenesis. However, the precise function of this factor in CNS development and the underlying molecular mechanisms are poorly understood. Here, we demonstrate that meteorin is involved in GFAP-positive glial differentiation through activation of the Jak-STAT3 pathway, by using neurosphere and retinal explant culture systems. During embryonic brain development, meteorin is highly expressed in neural stem and radial glia cells of the ventricular zone and immature neurons outside the ventricular zone but its expression disappears spontaneously as development proceeds except in GFAP-positive astrocytes. In cultured neurospheres, meteorin activates STAT3, and in turn increases the transcriptional activity of GFAP by enhancing the binding of STAT3 to the promoter. By meteorin stimulation, differentiating neurospheres show increased numbers of GFAP-positive cells, but the effect is abrogated by a blockade of the Jak-STAT3 pathway using either a Jak inhibitor or STAT3 siRNA. Furthermore, we expand our findings to the retina, and show that meteorin increases GFAP expression in Müller glia. Together, our results suggest that meteorin promotes GFAP-positive glia formation by mediating the Jak-STAT3 signaling pathway during both cortical stem cell differentiation and retinal glia development.

The Coxsackievirus–Adenovirus Receptor Reveals Complex Homophilic and Heterophilic Interactions on Neural Cells

The coxsackievirus-adenovirus receptor (CAR) is a member of the Ig superfamily strongly expressed in the developing nervous system. Our histological investigations during development reveal an initial uniform distribution of CAR on all neural cells with a concentration on membranes that face the margins of the nervous system (e.g., the basal laminae and the ventricular side). At more advanced stages, CAR becomes downregulated and restricted to specific regions including areas rich in axonal and dendritic surfaces. To study the function of CAR on neural cells, we used the fiber knob of the adenovirus, extracellular CAR domains, blocking antibodies to CAR, as well as CAR-deficient neural cells. Blocking antibodies were found to inhibit neurite extension in retina organ and retinal explant cultures, whereas the application of the recombinant fiber knob of the adenovirus subtype Ad2 or extracellular CAR domains promoted neurite extension and adhesion to extracellular matrices. We observed a promiscuous interaction of CAR with extracellular matrix glycoproteins, which was deduced from analytical ultracentrifugation experiments, affinity chromatography, and adhesion assays. The membrane proximal Ig domain of CAR, termed D2, was found to bind to a fibronectin fragment, including the heparin-binding domain 2, which promotes neurite extension of wild type, but not of CAR-deficient neural cells. In contrast to heterophilic interactions, homophilic association of CAR involves both Ig domains, as was revealed by ultracentrifugation, chemical cross-linking, and adhesion studies. The results of these functional and binding studies are correlated to a U-shaped homodimer of the complete extracellular domains of CAR detected by x-ray crystallography.

COUP-TFI and -TFII nuclear receptors are expressed in amacrine cells and play roles in regulating the differentiation of retinal progenitor cells

Chicken ovalbumin upstream promoter transcription factors (COUP-TFs) are members of the steroid/thyroid hormone receptor superfamily. We have shown that two homologous COUP-TF genes, COUP-TFI and COUP-TFII, are expressed in developing mouse retina with a unique gradient along the dorsal–ventral axis. In this work, we aimed to characterize the detailed expression patterns of COUP-TFs in mature retina. Their functions in retinal progenitor cell differentiation into subtypes of mature retinal cells were also examined. Immunostaining of frozen mouse retinal sections with antibodies against COUP-TFs and markers for retinal subtypes revealed that COUP-TFI and -TFII are expressed in amacrine cells, especially in a glycinergic subtype in mature mouse retina. Forced expression of COUP-TFI and -TFII in mouse retinal explant culture by retrovirus-mediated gene transfer promoted amacrine and cone photoreceptor cell differentiation, whereas that of rod photoreceptors decreased. Cell proliferation and apoptosis were not affected by the perturbation of COUP-TFI and -TFII expression levels. Using the Y79 retinoblastoma cell line, we observed that COUP-TFI and -TFII suppressed the transcriptional activation of the Nrl gene. We then analyzed one another member of COUP-TF transcription factors, COUP-TFγ, whose structure is relatively distant from those of COUP-TFI and -TFII. It is expressed mainly in horizontal cells and has weak activity in inducing amacrine cells when COUP-TFγ was ectopically expressed in retinal explants. In summary, we found that COUP-TFI and -TFII play roles in amacrine cell differentiation, and COUP-TFγ has distinct expression pattern and roles during retinal development.

Usingneurogeninto Reprogram Chick RPE to Produce Photoreceptor-like Neurons

One potential therapy for vision loss from photoreceptor degeneration is cell replacement, but this approach presents a need for photoreceptor cells. This study explores whether the retinal pigment epithelium (RPE) could be a convenient source of developing photoreceptors. The RPE of chick embryos was subjected to reprogramming by proneural genes neurogenin (ngn)1 and ngn3. The genes were introduced into the RPE through retrovirus RCAS-mediated transduction, with the virus microinjected into the eye or added to retinal pigment epithelial explant culture. The retinal pigment epithelia were then analyzed for photoreceptor traits. In chick embryos infected with retrovirus RCAS-expressing ngn3 (RCAS-ngn3), the photoreceptor gene visinin (the equivalent of mammalian recoverin) was expressed in cells of the retinal pigment epithelial layer. When isolated and cultured as explants, retinal pigment epithelial tissues from embryos infected with RCAS-ngn3 or RCAS-ngn1 gave rise to layers of visinin-positive cells. These reprogrammed cells expressed genes of phototransduction and synapses, such as red opsin, the alpha-subunit of cone transducin, SNAP-25, and PSD-95. Reprogramming occurred with retinal pigment epithelial explants derived from virally infected embryos and with retinal pigment epithelial explants derived from normal embryos, with the recombinant viruses added at the onset of the explant culture. In addition, reprogramming took place in retinal pigment epithelial explants from both young and old embryos, from embryonic day (E)6 to E18, when the visual system becomes functional in the chick. The results support the prospect of exploring the RPE as a convenient source of developing photoreceptors for in situ cell replacement.

Distribution of Müller stem cells within the neural retina: Evidence for the existence of a ciliary margin-like zone in the adult human eye

Much interest has been generated by the identification of neural stem cells in the human neural retina and ciliary body. However, it is not clear whether stem cells identified in these ocular compartments are of the same origin or whether they ontogenically derive from different cell populations. This study examined the in situ anatomical distribution of these cells within the neural retina and ciliary body, as well as their ability to proliferate in response to EGF. Human retinae and ciliary body were examined for co-expression of Nestin, cellular retinaldehyde binding (CRALBP) or Vimentin, and the stem cell markers SOX2, CHX10, NOTCH1 and SHH. Retinal explants were cultured with epidermal growth factor (EGF) to assess retinal cell proliferation. Intense Nestin and CRALBP staining was observed in the neural retinal margin, where cells formed bundles of spindle cells (resembling glial cells) that lacked lamination and co-stained for SOX2, CHX10 and SHH. This staining differentiated the neural retina from the ciliary epithelium, which expressed SOX2, CHX10 and NOTCH1 but not Nestin or CRALBP. Nestin and CRALBP expression decreased towards the posterior retina, where it anatomically identified a population of Müller glia. All Vimentin positive Müller glia co-stained for SOX2, but only few Vimentin positive cells expressed Nestin and SOX2. Cells of the retinal margin and the inner nuclear layer (INL), where the soma of Müller glia predominate, re-entered the cell cycle upon retinal explant culture with EGF. Lack of lamination and abundance of Müller glia expressing stem cell markers in the marginal region of the adult human retina resemble the ciliary marginal zone (CMZ) of fish and amphibians. The findings that cells in this CM-like zone, as well in the inner nuclear layer proliferate in response to EGF suggest that the adult human retina has regenerative potential. Identification of factors that may promote retinal regeneration in the adult human eye would provide efficient treatments for retinal degenerative conditions for which treatments are not yet available.

Transplantation prospects for the inner retina

Transplantation of stem or progenitor cells is an attractive new approach for treating neurodegenerative conditions of the central nervous system, which aims to protect or replace neurons and improve function. Proof of principle has already been shown in the retina that photoreceptors may be replaced by transplantation of neural progenitor cells. However, the task of retinal ganglion cell replacement is much more complex, as new cells will need to establish complex connections within the retina and also extend axons to precise targets in the brain. Although progress has been made in this field, it is likely that neuroprotective clinical applications will be established more quickly. Our laboratory has focused on the intraocular transplantation of cells to treat inner retinal disease, either by neuronal replacement or neuroprotection of existing cells. We have investigated the efficacy and effects of transplanting a variety of cell types, including human Müller stem cells (MIO-M1), oligodendrocyte precursor cells (OPCs), and bone marrow-derived mesenchymal stromal cells (MSCs) in a rat model of experimentally induced glaucoma. We also have developed and characterized a novel in vitro organotypic retinal explant culture system for exploring the methods of enhancing the efficacy of cell transplantation for the inner retina. In this review, we discuss the potentially beneficial effects of intraocular cell injections, identify current shortcomings of retinal stem cell therapy, and suggest directions for future research.

Involvement of retinoic acid signaling in goldfish optic nerve regeneration

Recently, we identified a retina-specific retinol-binding protein, purpurin, as a trigger molecule in the early stage of goldfish optic nerve regeneration. Purpurin protein was secreted by photoreceptors to injured ganglion cells, at 2–5 days after optic nerve injury. Purpurin bound to retinol induced neurite outgrowth in retinal explant cultures and retinoic acid (RA) had a comparable effect on neurite outgrowth. These results indicate that purpurin acts as a retinol transporter and facilitates conversion of retinol to RA. Intracellularly, RA is transported into the nucleus with cellular retinoic acid-binding protein IIb (CRABPIIb) and binds with retinoic acid receptor α (RARα) as a transcriptional regulator of target genes. Here, we investigated the RA signaling through RA synthesis to RARα in the goldfish retina during optic nerve regeneration by RT-PCR. Retinaldehyde dehydrogenase 2 (RALDH2; an RA synthetic enzyme) mRNA was increased by 2.7-fold in the retina at 7–10 days and then gradually decreased until 40 days after nerve injury. In contrast, cytochrome P450 26a1 (CYP26a1; an RA degradative enzyme) mRNA was decreased to less than half in the retina at 5–20 days and then gradually returned to the control level by 40 days after nerve injury. CRABPIIb mRNA was increased by 1.5-fold in the retina at 10 days after axotomy, RARαa mRNA was increased by 1.8-fold in the retina at 10 days after axotomy. The cellular changes in the RA signaling molecules after optic nerve injury were almost all located in the ganglion cells, as evaluated by in situ hybridization. The present data described for the first time that RA signaling through RALDH2 and CRABPIIb to RARα was serially upregulated in the ganglion cells at 7–10 days just after the purpurin induction. Therefore, we conclude that the triggering action of purpurin on optic nerve regeneration is mediated by RA signaling pathway.

Proneural gene ash1 promotes amacrine cell production in the chick retina

The diverse types of neurons and Müller glia in the vertebrate retina are believed to arise from common progenitor cells. To better understand how neural diversity is achieved during retinal neurogenesis, we examined the function of ash1, a proneural bHLH gene expressed in progenitor cells throughout retinal neurogenesis. Published studies using retinal explant culture derived from knockout mice concluded that ash1 is required for the production of late-born neurons, including bipolar cells. In this study, gain-of-function experiments were carried out in ovo in embryonic chick retina. In the developing chick retina, expression of ash1 temporally overlapped with, but spatially differed from, the expression of ngn2, also a proneural gene expressed in progenitor cells throughout retinal neurogenesis. Retrovirus-driven overexpression of ash1 in the developing chick retina decreased the progenitor population (BrdU+ or expressing ngn2), expanded the amacrine population (AP2alpha+ or Pax6+), and reduced bipolar (chx10 mRNA+) and Müller glial (vimentin+) populations. Photoreceptor deficiency occurred after the completion of neurogenesis. The number of ganglion cells, which are born first during retinal neurogenesis, remained unchanged. Similar overexpression of ngn2 did not produce discernible changes in retinal neurogenesis, nor in ash1 expression. These results suggest that ash1 promotes the production of amacrine cells and thus may participate in a regulatory network governing neural diversity in the chick retina.

TGIF, a homeodomain transcription factor, regulates retinal progenitor cell differentiation

TG-interacting factor (TGIF) is a TALE homeodomain protein expressed predominantly in the central nervous system and functions as a transcriptional repressor. Several mutations in TGIF have been identified in patients with holoprosencephaly, the most common congenital malformation of the developing human forebrain. However, the precise role of TGIF in neural development is not well understood. We found that TGIF was expressed strongly in the mouse retina during early stages of development, and that its expression gradually decreased as retinal development progressed. In vitro explant cultures of mouse retina mimic the in vivo development of retinal subtypes. Forced expression of TGIF using a retrovirus in explant culture induced the differentiation of amacrine cells from retinal progenitor cells. A TGIF paralog, TGIF2, showed a similar transition in expression during retinal development, and TGIF2 also promoted amacrine cell differentiation in a retinal explant culture system. However, no apparent difference between wild-type and TGIF-knockout mouse retina was observed, suggesting that TGIF and TGIF2 function redundantly in that tissue. Forced expression of TGIF homeodomain (HD)-EnR (repressing) rather than TGIF HD-VP16 (activating) resulted in a phenotype similar to that induced by wild-type TGIF, suggesting that TGIFs may act as transcriptional repressors to induce amacrine genesis.

Sox2 Plays a Role in the Induction of Amacrine and Müller Glial Cells in Mouse Retinal Progenitor Cells

The transcription factor Sox2 plays important roles in both human and mouse retinal development. Although loss-of-function mutations in Sox2 have been studied in mice, gain-of-function experiments in the neural retina have been lacking. The detailed expression pattern of Sox2 in the developing mouse retina was examined by immunohistochemistry. Then, Sox2 was expressed in a retinal explant culture prepared from E17 mouse embryos by retrovirus-mediated gene transfer to examine its role in retinal development. In addition, shRNA was used to suppress Sox2 in a retinal explant using a retrovirus-mediated system. Sox2 was expressed throughout the neuroblastic layer in the embryonic retina, but only in the inner nuclear layer in the mature retina. Double immunostaining revealed that Sox2 was expressed in Müller glial cells and in a subset of amacrine cells. Forced expression of Sox2 in a mouse retinal explant culture resulted in the dramatic accumulation of amacrine cells in the inner nuclear layer; in addition, cells expressing amacrine cell markers were also found on the innermost side of the outer nuclear layer. The expression of Pax6, which plays an important role in amacrine cell differentiation, was observed in the Sox2-expressing cells, and Sox2 activated the Pax6 promoter to drive luciferase expression in Y79 cells. A decrease in retinal progenitor cell proliferation accompanied these effects. The suppression of Sox2 expression by shRNA resulted in a decreased number of cells in the inner nuclear layer. Therefore, ectopic Sox2 expression can induce amacrine cells in the mouse retina from stage E17 onward, possibly by facilitating cell cycle exit.

Progenitor cell-derived factors enhance photoreceptor survival in rat retinal explants

Explantation of postnatal rat retinas is associated with degenerative events that show morphological similarities to human retinal degenerative disorders. The most evident morphological features are photoreceptor apoptosis involving caspase-3 and Müller cell activation. The purpose of the present study was to determine the content of protective factors in rat retinal progenitor cells and analyze the influence of the identified factors on the survival of photoreceptor cells and retinal gliosis. Tissue inhibitors of matrix metalloproteinase-1 (TIMP-1) and vascular endothelial growth factor (VEGF) were identified as putative beneficial factors, and their combined effect was examined in rat retinal explant cultures. Photoreceptor apoptosis was estimated by cell counts of cleaved caspase-3 and caspase-12 immunolabeled as well as TUNEL labeled cells. TIMP-1 and VEGF in combination significantly suppressed photoreceptor apoptosis involving caspase-3 activation. Cell counts of caspase-12 and TUNEL labeled photoreceptors showed no significant difference between the experiment and control retinas. TIMP-1 and VEGF appeared to have no effect on Müller cell activation as measured by GFAP and Ki-67 immunohistochemistry. Our data suggest that TIMP-1 and VEGF in combination promote the survival of photoreceptor cells in rat retinal explants, possibly by affecting a caspase-3 signaling pathway.

Development and Characterization of an Adult Retinal Explant Organotypic Tissue Culture System as an In Vitro Intraocular Stem Cell Transplantation Model

To develop and characterize a retinal explant culture system to facilitate investigation of novel methods of improving retinal stem cell therapy. Retinas explanted from adult rats were cultured in serum-free medium (B27/N2) or medium containing normal horse serum (NHS). Tissue viability was assessed by gross morphology, propidium iodide (PI) uptake, cell survival quantification, activated caspase-3 expression, and immunohistochemistry. Müller progenitor cells (hMIO-M1), or mesenchymal stem cells (MSC) were placed on explants, to model intravitreal cell transplantation. Explants were compared with whole eyes, with or without experimental glaucoma and/or intravitreal cell transplantation. Explants cultured in B27/N2 medium were viable for at least 17 days, as assessed by the aforementioned parameters. NHS medium was associated with obvious tissue degradation, greater/more diffuse PI uptake, significant cell loss over time, and temporal increase in activated caspase-3(+) cells. Explants in B27/N2 medium strongly expressed beta-III-tubulin, neurofilament, NeuN, Brn3a, Thy-1, GFAP, vimentin, nestin, and glutamine synthetase, whereas immunoreactivity was weak in NHS medium and decreased further with time. Seven and 14 days after coculture or transplantation, glial reactivity (GFAP/vimentin expression) was highly upregulated in explants and eyes, respectively. Some grafted cells migrated into the retina, but most remained outside the inner limiting membrane. Retinal explants prepared using the described techniques and cultured in B27/N2 medium are viable for at least 2 weeks and mimic in vivo glial reactivity to transplantation while allowing few grafted cells to integrate. This system may be a useful in vitro model for investigating methods of enhancing retinal stem cell therapy.

MARCKS-like protein, a membrane protein identified for its expression in developing neural retina, plays a role in regulating retinal cell proliferation

Membrane proteins are expressed in a specific manner in developing tissues, and characterization of these proteins is valuable because it allows them to be used as cell surface markers. Furthermore, they are potentially important for the regulation of organogenesis because some may participate in signal transduction. In the present study, we used proteomics to examine the comprehensive protein expression profile of the membrane fraction in the embryonic and adult mouse retina. We purified the retinal membrane fraction by sucrose-density-gradient centrifugation and analysed total proteins using shotgun analysis on a nanoflow LC-MS/MS (liquid chromatography tandem MS) system. Approximately half of the 326 proteins from the adult retina and a quarter of the 310 proteins from the embryonic retina (day 17) appeared to be membrane-associated proteins. Among these, MLP [MARCKS (myristoylated alanine-rich C-kinase substrate)-like protein], which shares approx. 50% amino acid identity with MARCKS, was selected for further characterization. The mRNA and surface protein expression of MLP decreased as retinal development progressed. Overexpression of MLP by retrovirus-mediated gene transfer enhanced the proliferation of retinal progenitor cells without affecting differentiation or cell migration in a retinal explant culture system. In contrast, MLP overexpression did not promote proliferation in fibroblasts (NIH 3T3 cells). Mutation analysis of MLP demonstrated that myristoylation was necessary to promote proliferation and that phosphorylation inhibited proliferation, indicating the functional importance of membrane localization.

Statistical analysis of data from retroviral clonal experiments in the developing retina

Retroviral lineage studies have been widely used over the past decade to study retinal development in vivo and in explant culture [Donovan S.L., Dyer, M.A., 2006. Preparation and Square Wave Electroporation of Retinal Explant Cultures, Nature Protocols 1, 2710–2718; Donovan, S.L., Schweers, B., Martins, R., Johnson D., Dyer, M.A., 2001. Compensation by tumor suppressor genes during retinal development in mice and humans, BMC Biol 4 , 14; Dyer M.A., Cepko, C.L., 2001. p27Kip1 and p57Kip2 regulate proliferation in distinct retinal progenitor cell populations, J. of Neurosci 21, 4259–4271; Dyer M.A., Cepko, C.L., 2000. p57(Kip2) regulates progenitor cell proliferation and amacrine interneuron development in the mouse retina, Development 127, 3593–3605; Dyer, M.A., Livesey, F.J., Cepko C.L., Oliver, G., 2003. Prox1 function controls progenitor cell proliferation and horizontal cell genesis in the mammalian retina, Nat Genet 34, 53–58]. These approaches can provide important data on the proliferation, cell fate specification, differentiation and survival of individual neurons and glia derived from single infected retinal progenitor cells. In some experiments, these parameters are compared in retinae from animals with different targeted deletions or transgenes. Alternatively, the effect of ectopic expression of virally encoded transgenes may be studied at the level of individual retinal progenitor cells in vivo and in explant culture. One of the challenges with interpreting retroviral lineage studies is determining the statistical significance of differences in the proliferation, cell fate specification, differentiation of survival of retinal progenitor cells between experimental and control samples. In this study, we provide a clear step-by-step guide to the application of statistical methods to retroviral lineage analyses actual data sets. We anticipate that this will serve as a guide for future statistical analyses of retroviral lineage studies and will help to provide a uniform standard in the field.

Early downregulation of IGF-I decides the fate of rat retinal ganglion cells after optic nerve injury

Retinal ganglion cells (RGCs) die by apoptosis after optic nerve injury. A number of reports have separately shown changes in pro-apoptotic proteins such as the Bcl-2 family members following optic nerve injury. However, induction time of these apoptotic signals has not been identified due to different treatments of the optic nerve, and insufficient time intervals for measurements. Therefore, the stream of cell death signals is not well understood. In the present study, we systematically reinvestigated a detailed time course of these cell death/survival signals in the rat retina after optic nerve crush, to determine the signal cascade leading to RGC apoptosis. The most conspicuous changes detected in the retina were the rapid inactivation of phospho-Akt and phospho-Bad proteins 2–3 days after optic nerve damage, and the subsequent gradual activation of Bax protein and caspase-3 activity accompanied by cell loss of RGCs 6 days after nerve injury. Cellular localization of these molecular changes was limited to RGCs. Furthermore, amount of insulin-like growth factor-I (IGF-I), an activator of the phosphatidyl inositol-3-kinase (PI3K)/Akt system, was initially decreased from RGCs 1–2 days just prior to the inactivation of phospho-Akt by optic nerve crush. Conversely, supplementation with IGF-I into the rat retina induced upregulation of phospho-Akt expression and cell survival of RGCs both in vitro and in vivo. Thus, injury to the optic nerve might induce early changes in cellular homeostasis with a plausible loss of trophic support for injured RGCs. Actually, IGF-I drastically enhanced neurite outgrowth from adult rat RGCs via a wortmannin-dependent mechanism in a retinal explant culture. Our data strongly indicate that IGF-I is a key molecule that induces RGC apoptosis or RGC survival and regeneration in the retina during the early stage of optic nerve injury.

Requirement of histone deacetylase activity for the expression of critical photoreceptor genes

BackgroundHistone deacetylases (HDACs) play a major role in the regulation of gene transcription, often leading to transcriptional repression, as well as other effects following deacetylation of non-histone proteins.ResultsTo investigate the role of HDACs in the developing mammalian retina, a general inhibitor of HDACs, trichostatin-A (TSA), was used to treat newborn murine retinae in explant cultures. Inhibition of HDAC activity resulted in a reduction in RNA levels for genes that regulate retinal development, as well as cell cycle regulators. Several of the genes encode transcription factors essential for rod photoreceptor development, Otx2, Nrl, and Crx. Using luciferase reporter assays, the promoter activity of both Nrl and Crx was found to be compromised by HDAC inhibition. Furthermore, downregulation of gene expression by HDAC inhibition didn't require de novo protein synthesis, and was associated with hyperacetylation of histones and non-histone proteins. Finally, HDAC inhibition in retinal explant cultures resulted in increased cell death, reduction in proliferation, a complete loss of rod photoreceptors and Müller glial cells, and an increase in bipolar cells.ConclusionHDAC activity is required for the expression of critical pro-rod transcription factors and the development of rod photoreceptor 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.

Activation of Multiple Pathways during Photoreceptor Apoptosis in therdMouse

The primary purpose of this study was to characterize photoreceptor apoptosis in the rd mouse. Given that apoptosis is the final common pathway in many cases of retinal degeneration, the ability to retard or even arrest this process may ameliorate retinal disorders such as retinitis pigmentosa (RP). The absence of any recognized therapy emphasizes the fact that a detailed knowledge of the molecular events involved is necessary to identify rational targets for therapeutic intervention. Flow cytometry was used to measure physical and chemical characteristics in the photoreceptor population. Individual cells flow in suspension past one or more lasers, scattering light and emitting fluorescence. Western blot techniques demonstrated cleavage of calpain-specific substrates. Retinal explant cultures were used for inhibitor studies. Postnatal day 10 (P(10)) rd retinas were cultured without retinal pigment epithelium (RPE) attached up to P(17). This study demonstrated calcium overload in the cytosol and subsequently in mitochondria. Mitochondrial membrane depolarization and reactive oxygen species (ROS) were detected later, during the peak of cell death. Analysis of downstream events indicated early activation of calcium-activated calpains. Treatment of rd retinal explants with the calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO (ALLN) successfully inhibited calpain-induced alpha-fodrin cleavage, yet it did not protect against photoreceptor degeneration. Finally, the results demonstrate an increase in the levels of both precursor and processed forms of the aspartate protease cathepsin D. Excessive calcium influx is an early event that initiates the activation of calcium-activated proteases. However, these proteases are not singularly the cause of death, because their inhibition does not prevent apoptosis. Indeed, the results presented herein suggest that multiple pathways are involved and that each of these components may have to be addressed for cell death to be successfully inhibited.

Negative regulation of retinal-neurite extension by β-catenin signaling pathway

Although there have been many studies on the regulation of neurite extension in mouse brain, such a mechanism in neural retina has remained to be clarified. To delineate the role of Wnt signaling in retinal development, we used a retrovirus-vector-mediated expression system to express various mutants forms of Wnt signaling members in E17.5 mouse retinal explant cultures, which are an excellent system to examine retinal development in vitro. Expression of constitutively active beta-catenin or Lef-1 in the retinal cells resulted in failure of neurite extension, suggesting that beta-catenin negatively regulates neurite extension in the retina through Lef-1 transcriptional activity. However, proliferation and differentiation of retinal cells into mature retinal cells such as rod-photoreceptor cells and Muller glia cells were not affected by perturbation of the Wnt-Lef-1 pathway. As in retinal cells, activation of beta-catenin-Lef-1 signaling inhibited NGF-induced neurite extension in PC12 cells without affecting their proliferation. Interestingly, the Wnt-Lef-1 signaling pathway suppressed neurite extension without affecting Mek-1 signal activity, which is known to promote neurite extension. We found that MAPK was activated in retinal explant cultures, but that perturbation of MAPK signals did not affect neurite extension. Taken together, our data suggest that the Wnt pathway functions in proper neurite extension by opposing positive signals for promotion of neurite extension that are distinct from those of the MAPK pathway.