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Updated: Jun 24, 2025
Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay
Published on: October 18, 2016
Qualitative evaluations of reactive microglial heterogeneity in cultured porcine retina
Kjell Johansson1, Camilla Mohlin2
1Department of Science, Kristianstad University, Kristianstad, Sweden. kjell.johansson@hkr.se.
Abstract:
A late stage of several retinal disorders is retinal detachment, a complication that results in rapid photoreceptor degeneration and synaptic damage. The porcine retina is a favorable in vitro model for studies of the degenerative processes that follow retinal detachment. Photoreceptor degeneration and synaptic injuries develop rapidly in the cultured porcine retina and correlate with resident microglial cell transition into a reactive phenotype. In this in vitro study, we used retinas cultured for five days and analyzed reactive CD11b and Iba1 immunoreactive microglia that localized close to/within the synaptic outer plexiform layer (OPL) and in the outer nuclear layer (ONL). A subpopulation of the CD11b and Iba1immunoreactive microglia also expressed CD68 immunoreactivity on lysosomal membranes or as a diffuse cytoplasmic stain. Some CD68 immunoreactive microglia were juxtaposed to L/M-opsin immunoreactive cone photoreceptors in the ONL. CD11b and Iba immunoelectron microscopy further suggests the presence of a dark microglial phenotype in the degenerating cultured porcine retina. For immunoelectron microscopy, nickel-enhanced diaminobenzidine (DAB) staining resulted in clearly distinguished reaction products in the cytosol of dark microglia.
Insights
In retinal detachment models, cultured porcine retinas show rapid photoreceptor degeneration. Microglia become reactive, with some expressing CD68 and appearing as a "dark" phenotype, indicating cellular stress.
Area of Science:
- Ophthalmology
- Neuroscience
- Cell Biology
Background:
- Retinal detachment is a late-stage complication of retinal disorders, leading to rapid photoreceptor degeneration and synaptic damage.
- The porcine retina serves as a suitable in vitro model for studying these degenerative processes.
- Microglial activation is observed during retinal degeneration following detachment.
Purpose of the Study:
- To investigate the characteristics and phenotypes of microglia in a degenerating cultured porcine retina model.
- To analyze the association of reactive microglia with photoreceptor layers and identify specific microglial markers.
Main Methods:
- Cultured porcine retinas were analyzed after five days.
- Immunohistochemistry was used to detect CD11b, Iba1, and CD68 immunoreactivity in microglia.
- Immunoelectron microscopy was employed to further characterize microglial phenotypes.
Main Results:
- Reactive CD11b and Iba1 immunoreactive microglia were found in the outer plexiform layer (OPL) and outer nuclear layer (ONL).
- A subpopulation of these microglia expressed CD68, with some located near cone photoreceptors.
- Immunoelectron microscopy suggested a distinct "dark" microglial phenotype in the degenerating retina.
Conclusions:
- Cultured porcine retinas exhibit rapid microglial activation and degeneration following simulated detachment.
- The presence of CD68 and the "dark" microglial phenotype indicate lysosomal activity and cellular stress in response to degeneration.
- This model is valuable for studying microglial responses in retinal degenerative diseases.
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