Characterisation of ramified microglial cells: detailed morphology, morphological plasticity and proliferative capability.

Several cellular properties of brain microglia in the rat were investigated using both whole tissue and cultures of dissociated cerebral cortical cells. As revealed by thiamine pyrophosphatase histochemistry, tissue microglia possessed a highly distinctive cellular morphology. Stained microglia showed similar overall features of morphology and distribution in both preparations; however, the cells in culture displayed some slight differences from those of the tissue, including larger somata and less developed processes. Through studying living ramified cells in culture, both morphological plasticity as evidenced by patterned variations in soma size and mitotic activity were directly confirmed. It was concluded that ramified microglia definitely possess proliferative capability, and this may reduce the need for blood cell recruitment in brain immune responses. In addition, cultured microglia exist in a somewhat more activated state than those in normal tissue, and in some instances undergo further activation as macrophages. This cortical tissue culture system should provide an amenable preparation for investigating the regulation of microglial function.

Dynamic features of cells expressing macrophage properties in tissue cultures of dissociated cerebral cortex from the rat.

Two previously identified forms of macrophage were investigated in primary cultures of cerebral cortical cells. Dynamic features were revealed through time-lapse video recording and aspects of macrophage function were assessed. The two cell forms were shown to be different pre-mitotic stages of a single cell type. The cell cycle for these cells involved an initial large, flat, quiescent cell which retracted to yield a slightly rounded form with numerous processes. This latter form lost processes and developed profuse filopodia as it became very rounded just prior to division; both resulting daughter cells then regained the initial large flat appearance. These cells possessed several properties of macrophages, including phagocytosis, nucleoside diphosphatase enzyme, and CR3 receptors. These properties were transient, expressed just before and after mitosis, but subsequently down-regulated in the flat daughter cells. Because of this feature, it was difficult to determine the exact size of this cell population; however, the observed rate of proliferation suggests it may be substantial. It is suggested that these cells correspond to non-microglial macrophages of brain tissue and, because of their significant down-regulation, they may be difficult to detect. This may be important in studies of brain accessory immune cells in tissue culture.

Characterization of ramified microglia in tissue culture: pinocytosis and motility.

Functional properties of ramified microglia were investigated in primary cultures of rat cerebral cortical cells. These microglia could be readily identified in both fixed and living cultures through previously established features. Based on their destruction by 5 mM L-leucine methyl ester, a high level of intrinsic endocytotic activity was established. When cultures were incubated with fluorescent latex beads to assess phagocytosis, little or no such activity was exhibited by ramified cells. However, when cultures were incubated with dyes or other soluble tracer compounds, these cells always exhibited labeling. This labeling was selective for ramified microglia in the cultures and was demonstrated using a variety of compounds, including trypan blue, lucifer yellow, horseradish peroxidase (HRP), and India ink. Intracellular label could be observed in vesicular structures; this localization corresponded to an active cellular process. Also, cellular labeling was inhibited by the presence of colchicine. These features supported the inference that the labeling was attributable to pinocytosis, and this process appeared to account for the vast majority of endocytotic activity in the ramified microglia. Possible physiological significance of this pinocytotic activity was indicated by the accumulation of various neurotransmitters/modulators: gamma-aminobutyric acid and vasoactive intestinal polypeptide (VIP). Ramified cells in these cultures have been previously noted to exhibit a constant and rapid pattern of motility, which was consistently observed here through time-lapse video recording; pinocytosis and rapid motility were shown to concur in individual cells. Based on their high intrinsic pinocytotic activity and pattern of cellular motility, the ramified microglia specifically are suggested to serve a constitutive function of fluid cleansing within the interstitial spaces of brain tissue.

Evidence for motility and pinocytosis in ramified microglia in tissue culture.

Ramified microglial cells were investigated in primary cultures of dissociated cerebral cortical tissue from rats. The identification of these cells was confirmed through immunohistochemical staining with 7 monoclonal antibodies selective for microglia. While there was significant variation in staining intensity with different antibodies, all stained the identified ramified cells; the antibodies OX-42 and ED1 yielded the most intense immunoreactivity. Based on distinctive morphological features, the microglia could be identified in living cultures where they were monitored using time-lapse video recording. This technique revealed extremely dynamic features of cellular plasticity and motility. Ramified microglia exhibited constant and rapid alterations in the size and shape of their cell body with an associated extension and retraction of processes; concomitantly, the cells moved about in a circumscribed area. These features of plasticity and motility were unique to this cell type, and correlated with OX-42 immunostaining. The microglia also possessed a differentially high level of pinocytotic activity; this too was correlated with OX-42 staining. From the nature of their morphological plasticity and motility, high pinocytosis, and cellular distribution, it is hypothesized that the ramified microglia specifically function as a system of fluid cleansing in normal brain tissue.

Pinocytotic activity in ramified microglia.

Pinocytotic activity was investigated in rat cerebral cortex using the soluble tracers horseradish peroxidase and Lucifer yellow. A subpopulation of cells selectively accumulated both compounds and the labelling was mainly present in pinocytotic vesicles associated with the cell body. Labelled cell bodies were small, round to oval, and distributed in an almost regular array throughout the tissue. Based on distinctive morphological features, some of the labelled cells could be determined as ramified microglia. This identification was confirmed by immunofluorescence staining with the monoclonal antibody OX-42, which specifically recognizes microglia; OX-42 staining consistently co-localized with pinocytotic labelling. The possibility that ramified microglial cells perform a normal function of continuous fluid exchange in brain tissue is discussed.

Method for the identification of brain macrophages/phagocytic cells in vitro.

The use of labelled latex beads, with acute incubation conditions, for the identification of active macrophages in mixed cell cultures based on their phagocytic activity is described. Fluorescent beads provided the best results and selectively labelled active macrophage cells in cultures of blood monocytes. When this technique was applied to primary cultures of rat cerebral cortex, a specific cell type was significantly labelled. This was a small round cell, previously uncharacterized, which in addition to phagocytic activity indicated by the ingestion of beads also possessed macrophage biochemical markers. Thus, the procedure appears useful for phagocytic macrophage identification in vitro, and should be generally applicable to any tissue culture system. Additionally, this procedure can be rendered compatible with cell viability and, therefore, be utilized for long-term monitoring of macrophages.