Microglia change from a reactive to an age-like phenotype with the time in culture

Abstract

Age-related neurodegenerative diseases have been associated with chronic neuroinflammation and microglia activation. However, cumulative evidence supports that inflammation only occurs at an early stage once microglia change the endogenous characteristics with aging and switch to irresponsive/senescent and dystrophic phenotypes with disease progression. Thus, it will be important to have the means to assess the role of reactive and aged microglia when studying advanced brain neurodegeneration processes and age-associated related disorders. Yet, most studies are done with microglia from neonates since there are no adequate means to isolate degenerating microglia for experimentation. Indeed, only a few studies report microglia isolation from aged animals, using either short-term cultures or high concentrations of mitogens in the medium, which trigger microglia reactivity. The purpose of this study was to develop an experimental process to naturally age microglia after isolation from neonatal mice and to characterize the cultured cells at 2 days in vitro (DIV), 10 DIV, and 16 DIV. We found that 2 DIV (young) microglia had predominant amoeboid morphology and markers of stressed/reactive phenotype. In contrast, 16 DIV (aged) microglia evidenced ramified morphology and increased matrix metalloproteinase (MMP)-2 activation, as well as reduced MMP-9, glutamate release and nuclear factor kappa-B activation, in parallel with decreased expression of Toll-like receptor (TLR)-2 and TLR-4, capacity to migrate and phagocytose. These findings together with the reduced expression of microRNA (miR)-124, and miR-155, decreased autophagy, enhanced senescence associated beta-galactosidase activity and elevated miR-146a expression, are suggestive that 16 DIV cells mainly correspond to irresponsive/senescent microglia. Data indicate that the model represent an opportunity to understand and control microglial aging, as well as to explore strategies to recover microglia surveillance function.

Activation and function of murine primary microglia in the absence of the prion protein

“…Phagocytic activity of microglia was therefore addressed in an assay of uptake of zymosan-FITC bioparticles. Surprisingly, although the basal phagocytic activity of microglial cells was high -as expected for young primary microglial cultures (Caldeira et al, 2014) -there was no sign of an effect of the microglia genotype in the uptake of zymosan before or after LPS treatment (Fig. 6).…”

MicroRNA-146a: A Dominant, Negative Regulator of the Innate Immune Response

“…Interestingly, the levels of miR-146a have also been shown to increase in aged microglia as well as macrophages and have been implicated in age-related dysfunction of macrophages (88, 89). Macrophages from aging mice exhibited a lack of response to stimulation with LPS and pro-inflammatory cytokines thus interrupting the negative feed-back regulation loop regulated by miR-146a.…”

Potassium channel expression and function in microglia: Plasticity and possible species variations

“…Additionally, microglia senescence during aging or in prolonged in vitro culture can alter their responsiveness to stimuli. 46 Thus, particular attention should be paid to the age of the animals, as well as to the length of time, microglia are kept in culture for differentiation studies to avoid unintentional skewing of microglia activation and the resulting K C channel expression. 2) Acutely isolated microglia cells and slices from animal models: Functional K C channel expression in microglia should be studied more thoroughly in both mouse and rat models of diseases accompanied by neuroinflammation, such as ischemic stroke, Alzheimer disease, Parkinson disease, traumatic brain injury, and epilepsy.…”