Abstract
Microglia are the principal glial cells involved in the processes of immune inflammation within both retina and optic nerve, especially under the context of glaucomatous neuropathy. Considering the distinguishing role of retinal microglia in glaucoma and the lack of established protocol for microglia isolation from animal glaucoma model, the present study aimed to develop and validate a method with characteristics of both simplicity and efficiency for retinal microglia isolation from chronic ocular hypertensive (COH) rats. A Percoll gradient of various concentrations was used to separate microglia from whole retinal cells of the COH rats and control group. The finally isolated microglia were identified by CD11b and Iba-1 immunofluorescence staining, and the cell viability was determined by trypan blue staining. Additionally, the proportion of microglia in the whole retina cells was identified by flow cytometry. Results showed that the survival rates of isolated retinal microglia with the Percoll gradient method were 67.2 ± 4% and 67.6 ± 3% in control and COH groups, respectively. The proportion of the microglia population in the whole retinal cells was about 0.4–0.93%. To conclude, the present study confirmed that the application of Percoll gradient could effectively separate microglia from retinas of COH rats, which will probably enrich the tool kit for basic researchers of glaucoma specialty and help with scientific investigations.
Highlights
Microglia are macrophages located in the brain and spinal cord as well as in the retina and therein act as innate immune cells
The cells separated from the retina of the chronic ocular hypertensive (COH) rat with the Percoll gradient concentration method were resuspended in 1 mL of Hanks’ balanced salt solution (HBSS)
The amount of primary retinal microglia isolated from the COH rat and the control group using the Percoll gradient
Summary
Microglia are macrophages located in the brain and spinal cord as well as in the retina and therein act as innate immune cells. M1 phenotypic cells usually refer to the microglia, which express pro-inflammatory cytokines (iNOS, TNF-α, IL-1, IL-6, etc.) if stimulated by interferon-γ or TNF-α [7,8]. Dick et al only used flow cytometry to identify the antigen-presenting cells of normal rat microglia, and some other research subjects were nonpathological dogs [25–27] This experimental tool has not yet been applied to retinas of the glaucoma model, such as rats of induced chronic ocular hypertension. Considering the acknowledged essential role of microglia in the process of glaucomatous neuropathy, and with the aim to facilitate characterizing microglia immunophenotypes and function in the context of glaucoma as well as other relevant retinal diseases, we attempted to isolate the microglia cells from COH rat retinas using the protocol of Percoll concentration gradient centrifugation. The Percoll concentration gradient method was applied to isolate retinal microglia in the COH rat model and provide a new and effective method to facilitate the study of retinal microglia in glaucoma as well as other ocular diseases
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