Article

Photoreceptor vitality in organotypic cultures of mature vertebrate retinas validated by light-dependent molecular movements

Institute of Zoology, Department of Cell and Matrix Biology, University of Mainz, Germany.
Vision Research (Impact Factor: 2.38). 01/2007; 46(27):4464-71. DOI: 10.1016/j.visres.2006.07.019
Source: PubMed

ABSTRACT Vertebrate photoreceptor cells are polarized neurons highly specialized for light absorption and visual signal transduction. Photoreceptor cells consist of the light sensitive outer segment and the biosynthetic active inner segment linked by a slender connecting cilium. The function of mature photoreceptor cells is strictly dependent on this compartmentalization which is maintained in the specialized retinal environment. To keep this fragile morphologic and functional composition for further cell biological studies and treatments we established organotypic retina cultures of mature mice and Xenopus laevis. The organotypic retina cultures of both model organisms are created as co-cultures of the retina and the pigment epithelium, still attached to outer segments of the photoreceptor cells. To demonstrate the suitability of the culture system for physiological analyses we performed apoptotic cell death analyses and verified photoreceptor viability. Furthermore, light-dependent bidirectional movements of arrestin and transducin in photoreceptors in vivo and in the retinal cultures were indistinguishable indicating normal photoreceptor cell-biologic function in organotypic cultures. Our established culture systems allow the analysis of mature photoreceptor cells and their accessibility to treatments, characteristic for common cell culture. Furthermore, this culturing technique also provides an appropriate system for gene delivery to retinal cells and will serve to simulate gene therapeutic approaches prior to difficult and time-consuming in vivo experiments.

Download full-text

Full-text

Available from: Tobias Goldmann, Aug 16, 2015
0 Followers
 · 
93 Views
  • Source
    • "Murine and human retinas were prepared as previously described (Goldmann et al, 2011; Reidel et al, 2006). For biocompatibility, PN10 retinas were incubated for 2 days. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Translational read-through-inducing drugs (TRIDs) promote read-through of nonsense mutations, placing them in the spotlight of current gene-based thera-peutic research. Here, we compare for the first time the relative efficacies of new-generation aminoglycosides NB30, NB54 and the chemical compound PTC124 on retinal toxicity and read-through efficacy of a nonsense mutation in the USH1C gene, which encodes the scaffold protein harmonin. This mutation causes the human Usher syndrome, the most common form of inherited deaf-blindness. We quantify read-through efficacy of the TRIDs in cell culture and show the resto-ration of harmonin function. We do not observe significant differences in the read-through efficacy of the TRIDs in retinal cultures; however, we show an excellent biocompatibility in retinal cultures with read-through versus toxicity evidently superior for NB54 and PTC124. In addition, in vivo administration of NB54 and PTC124 induced recovery of the full-length harmonin a1 with the same efficacy. The high biocompatibilities combined with the sustained read-through efficacies of these drugs emphasize the potential of NB54 and PTC124 in treating nonsense mutation-based retinal disorders.
    EMBO Molecular Medicine 11/2012; 4(11). DOI:10.1002/emmm.201201438 · 8.25 Impact Factor
  • Source
    • "Murine and human retinas were cultured as previously described (Reidel et al., 2006). Terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) was performed according to the protocol of the manufacturer (In Situ cell death detection kit; Roche, Mannheim, Germany). "
    [Show abstract] [Hide abstract]
    ABSTRACT: We investigated the therapeutic potential of the premature termination codon (PTC) readthrough-inducing drug PTC124 in treating the retinal phenotype of Usher syndrome, caused by a nonsense mutation in the USH1C gene. Applications in cell culture, organotypic retina cultures, and mice in vivo revealed significant readthrough and the recovery of protein function. In comparison with other readthrough drugs, namely the clinically approved readthrough-inducing aminoglycoside gentamicin, PTC124 exhibits significant better retinal biocompatibility. Its high readthrough efficiency in combination with excellent biocompatibility makes PTC124 a promising therapeutic agent for PTCs in USH1C, as well as other ocular and nonocular genetic diseases.
    Human gene therapy 05/2011; 22(5):537-47. DOI:10.1089/hum.2010.067 · 3.62 Impact Factor
  • Source
    • "The retina culture system was established according to the experimental procedures previously published by Reidel et al. (2006). In brief, intact eyes of adult Long–Evans rat were immediately removed from animals that had been killed and incubated with 1.2 mg ml −1 Proteinase K (Sigma-Aldrich) for 30 min at 37 • C. Proteinase K activity was stopped by transferring the eyes to the culture medium containing 10% fetal calf serum for 5 min. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Light adaptation in vertebrate photoreceptors is mediated by multiple mechanisms, one of which could involve nuclear feedback and changes in gene expression. Therefore, we have investigated light adaptation-associated changes in gene expression using microarrays and real-time PCR in isolated photoreceptors, in cultured isolated retinas and in acutely isolated retinas. In all three preparations after 2 h of an exposure to a bright light, we observed an up-regulation of almost 100% of three genes, Sag, Guca1a and Guca1b, coding for proteins known to play a major role in phototransduction: arrestin, GCAP1 and GCAP2. No detectable up-regulation occurred for light exposures of less than 1 h. Functional in vivo electroretinographic tests show that a partial recovery of the dark current occurred 1-2 h after prolonged illumination with a steady light that initially caused a substantial suppression of the photoresponse. These observations demonstrate that prolonged illumination results in the up-regulation of genes coding for proteins involved in the phototransduction signalling cascade, possibly underlying a novel component of light adaptation occurring 1-2 h after the onset of a steady bright light.
    The Journal of Physiology 04/2009; 587(Pt 11):2457-72. DOI:10.1113/jphysiol.2009.168609 · 4.54 Impact Factor
Show more