Morphological, immunocytochemical and functional characterization of the microglial phenotype in culture

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Selected morphological, immunocytochemical and functional aspects of various microglial cell populations were characterized in mixed neuronal/glial and pure microglial cultures. The mixed primary cortical cultures were prepared from the forebrains of embryonic (E18) rats and maintained for up to 28 days (DIV1–DIV28) using routine culturing techniques. The pure microglial cells were subcloned (subDIV4) from the mixed primary cultures and maintained for up to 7 days (DIV7). During culturing, expansion of the microglial cells was observed, as evidenced by quantitative assessment of selected monocyte/macrophage/microglial cellspecific markers (HLA DP, DQ, DR, CD11b/c and Iba1) via immunocyto- and histochemistry and Western blot analysis. The Iba1 immunoreactivity in Western blots steadily increased about 750-fold, and the number of Iba1-immunoreactive cells rose at least 67-fold between DIV1 and DIV28. Morphometric analysis on binary (digital) silhouettes of the microglia revealed their evolving morphology during culturing. Microglial cells were mainly ameboid in the early stages of in vitro differentiation, while mixed populations of ameboid and ramified cell morphologies were characteristic of older cultures as the average transformation index (TI) increased from 1.96 (DIV1) to 15.17 (DIV28). Multiple immunofluorescence labeling of selected biomarkers revealed different microglial phenotypes during culturing. For example, while HLA DP, DQ, DR immunoreactivity was present exclusively in ameboid microglia (TI < 3) between DIV1 and DIV10, CD11b/c- and Iba1-positive microglial cells were moderately (TI < 13) and progressively (TI < 81) more ramified, respectively, and always present throughout culturing. Regardless of the age of the cultures, proliferating microglia were Ki67- positive and characterized by low TI values (TI < 3). The microglial function was assessed by an in vitro phagocytosis assay. Unstimulated microglia with low TI values were significantly more active in phagocytosing fluorescent microspheres than the ramified forms. The roles of calmodulin (CaM), a multifunctional intracellular calcium receptor protein, as concerns selected morphological and functional characteristics of pure microglial cells were investigated through use of the CaM antagonists calmidazolium (CALMID) and trifluoperazine (TFP). The intracellular localization of the CaM protein relative to phalloidin, a bicyclic heptapeptide that binds only to filamentous actin, and the ionized calcium-binding adaptor molecule 1 (Iba1), a microglia-specific actin-binding protein, was determined by immunocytochemistry, with quantitative analysis by immunoblotting. In unchallenged and untreated (control) microglia, high concentrations of CaM protein were found mainly perinuclearly in ameboid microglia, while the cell cortex had a smaller CaM content that diminished progressively deeper into the branches in the ramified microglia. The amounts and 5 intracellular distributions of both Iba1 and CaM proteins were altered after lipopolysaccharide (LPS) challenge in activated microglia. CALMID and TFP exerted different, sometimes opposing, effects on many morphological, cytoskeletal and functional characteristics of the microglial cells. They affected the CaM and Iba1 protein expressions and their intracellular localizations differently, inhibited cell proliferation, viability and fluid-phase phagocytosis to different degrees both in unchallenged and in LPS-treated (immunologically challenged) cells, and differentially affected the reorganization of the actin cytoskeleton in the microglial cell cortex, influencing lamellipodia, filipodia and podosome formation. We concluded that in vitro studies on microglial population dynamics combined with phenotypic characterization can be of importance when different in vivo pathophysiological situations are modeled in vitro. Moreover, the CaM antagonists altered different aspects of filamentous actin-based cell morphology and related functions with variable efficacy, which could be important in deciphering the roles of CaM in regulating microglial functions in health and disease.

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