Culture and Examination of Neurotoxic and Vascular Permeability-inducing Potential of Microglia Obtained from Adult Rats

Abstract

Purpose: : To study the inflammatory potential, neurotoxicity, and effect on endothelial barrier permeability of activated microglia.

Methods: : Microglia are resident tissue macrophage-like cells that represent the innate immune system within central and peripheral nervous tissue. Microglia monitor their environment and upon detecting damage or infection switch from a resting to an activated state. Microglial activation results in the production of copious amounts of inflammatory cytokines, nitric oxide (NO•), and reactive oxygen species (ROS), which can cause retinal neurodegeneration and permeability of the blood-retinal barrier. Established methods of obtaining microglia utilize mixed glial cultures from neonatal rat brains. We developed a method to obtain cultures of resting microglia from adult rat neuronal tissue, thus allowing the study of retinal microglial activation. Using cell co-culture techniques and the transfer of microglia-conditioned media, the ability of resting and activated microglia to cause retinal neuronal cell death and endothelial layer permeability was examined.

Results: : Activated microglia caused caspase activation and death of R28 retinal neuronal cells, as well as increased permeability of junctional barriers formed by bovine retinal endothelial cells (BREC). This coincided with the microglial production of numerous pro-inflammatory molecules, including interleukin-1beta, tumor necrosis factor alpha, vascular endothelial growth factor and NO•. Anti-inflammatory agents, including the tetracycline derivatives doxycycline and minocycline, partially prevented the expression of inflammatory factors as well as the detrimental effects of activated microglia on R28 viability and BREC barrier integrity.

Conclusions: : Activated retinal microglia produce factors that can promote the death of retinal neurons and loss of blood-retinal barrier integrity. Anti-inflammatory agents that target the microglial response to activation have the potential to prevent retinal dysfunction by diminishing the production of these mediators.

Keywords: microglia • retinal degenerations: cell biology • inflammation