Ex vivo cultures of microglia from young and aged rodent brain reveal age-related changes in microglial function

Abstract

To understand how microglial cell function may change with aging, various protocols have been developed to isolate microglia from the young and aged central nervous system (CNS). Here we report modification of an existing protocol that is marked by less debris contamination and improved yields and demonstrate that microglial functions are varied and dependent on age. Specifically, we found that microglia from aged mice constitutively secrete greater amounts of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) relative to microglia from younger mice and are less responsive to stimulation. Also, microglia from aged mice have reduced glutathione levels and internalize less amyloid beta peptide (Aβ) while microglia from mice of all ages do not retain the amyloid beta peptide for a significant length of time. These studies offer further support for the idea that microglial cell function changes with aging. They suggest that microglial Aβ phagocytosis results in Aβ redistribution rather than biophysical degradation in vivo and thereby provide mechanistic insight to the lack of amyloid burden elimination by parenchymal microglia in aged adults and those suffering from Alzheimer's disease.

Fig. 1

Dispase II density centrifugation methodology: (A) Brain hemispheres that were homogenized with dispase II and loaded onto discontinuous gradients composed of 35% ‘low’ percoll and 75% ‘high’ percoll sequestered microglia to an isopynic density of 1.077mg/µl as determined by beads with known densities. This configuration spatially separated unwanted brain matter to a density 50µg/µl more buoyant. (B) Density centrifugation methodology as described in the literature involved mechanical homogenization and reduced percoll densities. In our hands, such methodology failed to channel unwanted brain matter (tissue chunks, red arrowhead) to an isopynic position distal to the microglia enriched band. (C) Phase contrast images of freshly prepared cells under a hemacytometer demonstrate reduced particulate matter from dispase II homogenized brains and viable phase-bright cells that exclude trypan blue. Following 24hrs of culture, these cells remain adherent after multiple washes (D) and are thus compatible with experiments that involve media exchanges.

Fig. 2

Purity, yield and viability of microglia: (A) Microglia isolated with dispase II density centrifugation methodology express Iba1, a marker commonly used to identify microglia in vivo. (B) Yields of microglia from 1 month old and 15 month old mice typically obtained using dispase II based density centrifugation methodology show little variability with age. (C) Measurement of mitochondrial respiratory activity indicated that isolated microglia form cultures comparable in viability to HEK 293 cells, an immortal cell line.

Fig. 3

Cytokine secretion of young and aged microglia: (A) Upon stimulation with the biological immunostimulatory agent LPS (100ng/ml), or Pam3CSK4, a synthetic agonist of toll-like receptor 2 (1µg/ml), IL-6 production by aged microglia was markedly increased when compared to young microglia. (B) LPS (100ng/ml) stimulated similar TNF-α production in microglia derived from young and aged mice. Yet, TNF-α production was significantly increased in aged microglia following Pam3CSK4 (1µg/ml) exposure. *, p<0.05; #, p<0.001.

Fig. 4

(A) Western blot analysis using 6E10, an antibody specific to the first 16 amino acids of Aβ42, indicates monomeric (4kDa), oligomeric (16kDa, 20kDa) and higher-order conformations larger than 220kDa in stock preparations as well as preparations that have been exposed to microglia. Higher-order conformations larger than 220kDa persist following the exposure of samples to buffer containing 2% SDS suggesting the presence of fibrillar species. 10% serum in media overloads gel at 60kDa and may block visibility of some Aβ42 species. 75ng Aβ42/well. (B) Internalization of Aβ42 by microglia from adult mice was directly observed in living microglia with FITC conjugated Aβ42 peptide and with 6E10 immunocytochemistry. In both cases, Aβ42 had a perinuclear localization. Lamp1 colocalization with 6E10 immunoreactivity suggests that some Aβ42 reached microglial lysosomal compartments (C).

Fig. 5

Fate of Aβ internalized by microglia: Microglia extracted from mice of various ages were exposed to Aβ42 preparations containing monomeric, oligomeric and SDS-resistant fibrillar species (reflecting in vivo amyloid diversity) in pulse-chase experiments. (A) Neonatal and young microglia respectively internalized 74% and 53% more Aβ42 relative to aged microglia. (B) Invariably, internalized Aβ42 was expelled by neonatal and young microglia within 3hrs of ingestion, suggesting disengagement from biophysical degradation following phagocytosis. Mock data (gray) represents experiments without the presence of cells to control for non-specific adherence of Aβ to culture wells. Detection of Aβ42 requires the presence of both NH₂ and COOH terminals of Aβ42, thus only intact Aβ42 peptides are quantified in the above experiments. *, p<0.05; **, p<0.01.