Viability of Mouse Retinal Explant Cultures Assessed by Preservation of Functionality and Morphology

Abstract

PURPOSE. Retinal explant cultures provide simplified systems where the functions of the retina and the effects of ocular therapies can be studied in an isolated environment. The purpose of this study was to provide insight into long-term preservation of retinal tissue in culture conditions, enable a deeper understanding of the interdependence of retinal morphology and function, and ensure the reliability of the explant technique for prolonged experiments. METHODS. Retinal explants from adult mice were cultured as organotypic culture at the airmedium interface for 14 days in vitro (DIV). Retinal functionality was assessed by multielectrode array technique and morphology by immunohistochemical methods at several time points during culture. RESULTS. Retinal explants retained viability for 14 DIV, although with diminishing neuronal activity, progressing neuronal loss, and increasing reactive gliosis. We recorded spontaneous retinal ganglion cell (RGC) activity up to 14 DIV with temporally changing distribution of RGC firing rates. Light responsiveness was measurable from RGCs for 7 DIV and from photoreceptors for 2 DIV. Apoptotic cells were detected beginning at 3 DIV with their density peaking at 7 DIV. The number of RGCs gradually decreased by 70% during 14 DIV. The change was accompanied by the loss of RGC functionality, resulting in 84% loss of electrically active RGCs. CONCLUSIONS. Retinal explants provide a valuable tool for studies of retinal functions and development of ocular therapies. However, critical for long-term use, retinal functionality was lost before structural loss, emphasizing a need for both functional and morphologic readouts to determine the overall state of the cultured retina.

Synchrony of Spontaneous Burst Firing between Retinal Ganglion Cells Across Species

“…RPE detachment from the retina can induce severe damage to the nearest photoreceptors (PRs) rather than the relatively distant RGCs. In some experiments of RPE detachment, after removal of RPE, the visual pathway from PR to RGC was functionally inactive while RGC's spontaneous firing was well maintained and robust [ 64 - 66 ]. To improve the tissue quality during the time window from enucleation to recording, we used a CO 2 -independent medium well known as transport buffer to avoid tissue damage caused by pH changes of the buffer solution.…”

Physiological assessment of high glucose neurotoxicity in mouse and rat retinal explants

“…In order to account for possible differential effects of diabetic conditions on the retinal ON and OFF pathways, an OFF BC type (BC‐3a) was analyzed in addition to RBCs after 7 and 14 days in culture. To our knowledge, only two studies have done an electrophysiological analysis in retinal explants (Alarautalahti et al, ; Koizumi et al, ). However, the ganglion cells analyzed in these studies have the disadvantage of suffering from variable degrees of retrograde degeneration due to loss of their axons in the explantation process.…”

“…Very little is known about the electrophysiological properties of retinal cells after weeks in organotypic culture, if they still reflect the properties of cells in the in vivo retina. Exceptions are one study in explants from adult rabbits cultured for 6 days, suggesting a preserved function of ganglion cells after this period (Koizumi, Zeck, Ben, Masland, & Jakobs, ), and another recent study in mouse explants, reporting a residual degree of activity and light responses in ganglion cells after 7 days in culture (Alarautalahti et al, ). However, ganglion cells are hampered as indicators of retinal integrity in explants because their axons have been severed, leading to retrograde degeneration independent from the culture conditions.…”

Structural engraftment and topographic spacing of transplanted human stem cell-derived retinal ganglion cells

“…73 Spontaneous electrophysiological activity in organotypic retinal explants is modest, decreases with time, and may have limited any potential IPL-directed dendrite localization reinforced by neuronal activity. 74 During development, RGC dendrites target pre-patterned IPL afferents. 75 Sublamination within the IPL is guided by the expression of specific cell surface receptors and their binding to localized lamina-specific ligands, including integrins, cadherins, plexins, and others play critical roles in dendritic outgrowth and guidance.…”