Organotypic Cultures of Adult Mouse Retina: Morphologic Changes and Gene Expression

• Published in: Investigative ophthalmology & visual science Vol. 58; no. 4; pp. 1930 - 1940
• Main Authors: Müller, Brigitte, Wagner, Franziska, Lorenz, Birgit, Stieger, Knut
• Format: Journal Article
• Language: English
• Published: United States 01.04.2017
• Subjects: Animals; Cell Culture Techniques; Cell Death; DNA - genetics; Follow-Up Studies; Gene Expression; Immunohistochemistry; In Situ Nick-End Labeling; Mice; Mice, Inbred C57BL; Neuroglia - metabolism; Neuroglia - pathology; Organ Culture Techniques; Polymerase Chain Reaction; Retina - metabolism; Retina - pathology; Retinal Degeneration - genetics; Retinal Degeneration - metabolism; Retinal Degeneration - pathology; Retinal Photoreceptor Cell Outer Segment - metabolism; Retinal Photoreceptor Cell Outer Segment - pathology; RNA, Messenger - genetics
• ISSN: 1552-5783; 1552-5783
• DOI: 10.1167/iovs.16-20718

The purpose of this study was characterization of adult murine neuroretina in organ culture to investigate its suitability for use in preclinical therapeutic applications. In retinal disorders, neurodegeneration of mature retinal cells takes place. Therefore, neonatal retina cultures are not adequate for therapeutic applications, such as genome editing, as the retina is still developing with cells dividing and differentiating into highly specialized cell types such as photoreceptors. Retinal explants were prepared from 3-month-old wild-type C57Bl6 mice and evaluated after 1 to 10 days in culture by immunohistochemistry or quantitative (q)PCR. Histologic modifications of the neuroretina were evaluated with TUNEL assay and immunohistochemical markers for neurons, glia cells, and apoptosis. During the first week, disruption and truncation of outer segments were detectable. Unspecific Müller cell reaction was detected from 4 days in culture. Sprouting of individual rod bipolar cell dendrites into the outer nuclear layer (ONL) was visible during all explant stages. During the second week in culture, cell death in the ONL became more prominent. Adult organotypic retinal culture was successful for at least 10 days with characteristic alterations of the morphology during this period. This characterization forms the basis to establish retinal explants for gene therapeutic applications as an intermediate step between cell culture and experiments on adult animals, thus reducing the load of animal experimentation.