Migration and ramification of microglia in quail embryo retina organotypic cultures

Abstract

Organotypic cultures of retina explants preserve the complex cellular microenvironment of the retina and have been used as a tool to assess the biological functions of some cell types. However, studies to date have shown that microglial cells activate quickly in response to the retina explantation. In this study, microglial cells migrated and ramified in quail embryo retina organotypic cultures (QEROCs) according to chronological patterns bearing a resemblance to those in the retina in situ, despite some differences in cell density and ramification degree. Retinal explants from quail embryos at 9 days of incubation (E9) proved to be the best in vitro system for reproducing a physiological-like behavior of microglial cells when cultured in Eagle's basal medium supplemented with horse serum. During the first week in vitro, microglial cells migrated tangentially in the vitreal part of QEROCs, and some began to migrate radially from 3 days in vitro (div) onward, ramifying in the inner and outer plexiform layers, thus mimicking microglia development in the retina in situ, although reaching a lower degree of ramification after 7 div. From 8 div onward, microglial cells rounded throughout the explant thickness simultaneously with the nonphysiological appearance of dead photoreceptors and round microglia in the outernuclear layer. Therefore, E9 QEROCs can be used during the first week in vitro as a model system for experimental studies of molecules putatively involved in microglial migration and ramification.

Expression of Inducible Nitric Oxide Synthase (iNOS) in Microglia of the Developing Quail Retina

“…1A), with an elongated cell body bearing two cell processes in opposite poles and broad lamellipodia, as previously described [54]. After LPS treatment for 24 hiv, microglia in E8 retina explants changed their morphological features, showing a more rounded cell body with scarce, shorter cell processes and infrequent lamellipodia (Figure 9A).…”

“…In the present study, DAR-4M AM was used in E8+12hiv retina explants to avoid technical problems inherent to its in vivo utilization. Given the physiologic behavior of microglial cells in E8 quail retina explants up to 24 hiv [54], microglial NO production in these explants would be representative of the production in vivo . The upregulation of iNOS expression in response to LPS treatment provides further indirect evidence of iNOS expression in normal retina amoeboid microglia.…”

“…E8 retina explants cultured for 12 hiv in the presence (5 µg/mL) or absence of LPS, as described above, were treated with 5 µM DAR-4M AM and 0.4 µg/ml Alexa Fluor-488 conjugated QH1 antibody (AF488-QH1) for 1 h at 37°C. AF488-QH1 marked microglial cells in the explants with strong green fluorescent labeling without affecting their physiological behavior [54], whereas DAR-4M AM labeled NO-producing cells with red fluorescence. After AF488-QH1 and DAR-4M AM treatment, the explants were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 45 min and coverslipped with Fluoromount G. Observations of wholemounted explants were made using a Zeiss Axiophot microscope (Zeiss, Oberkochen, Germany) equipped for epifluorescence, obtaining micrographs with a Zeiss AxioCam digital camera.…”

“…Explants from E8 retinas were cultured in vitro on 30-mm Millicell CM culture plate inserts (Millipore, Billerica, MA; pore size 0.4 µm) according to the method described by Stoppini et al (1991) [53] with some modifications [54]. Retinas were dissected out into cold Gey's balanced salt solution (Sigma, St. Louis, MO) supplemented with 5 mg/mL glucose (Sigma) and 50 IU-µg/mL penicillin-streptomycin (Invitrogen, Paisley, United Kingdom).…”

Expression of Metabolic Coupling and Adhesion Proteins in the Porcine Optic-Nerve Head: Relevance to a Flow Model of Glaucoma

“…Explants of the dissected prelaminar region of the optic nerve head of recently enucleated pig eyes were cultured in vitro following the method described by Stoppini et al (1991) as adapted by Carrasco et al (2010). Briefly, optic discs were dissected with a surgical blade in a sterile environment and placed in a Petri dish containing the same culture medium as detailed below.…”