Resting microglial cells in vitro: Analysis of morphology and adhesion molecule expression in organotypic hippocampal slice cultures

Abstract

Neurons in organotypic hippocampal slice cultures (OHSCs) are known to preserve morphological and physiological features of the in vivo situation; however, little is known about the properties of microglial cells under these in vitro conditions. In this study, we addressed the question whether microglial cells in OHSCs are initially activated following explantation but return to a resting state during in vitro cultivation. Thus, we analyzed a) microglial cell morphology, b) microglial cell distribution, and c) expression of integrin adhesion molecules as putative markers of microglial activation. Hippocampal slices fixed immediately following explantation showed only resting microglial cells, mainly located in the paraventricular regions. After 3 days in vitro (div) OHSC surfaces were covered by activated microglia, whereas intermediate layers contained fewer microglial cells, giving the slices a sandwich‐like appearance with the intact hippocampal formation being surrounded by glial tissue. After 3 div, microglial cells in intermediate layers of OHSCs showed activated morphology with ovaloid cytoplasm and no or merely few cytoplasmic processes; after 6 div, however, an increasing degree of ramification could be observed. After 9 div, microglia in intermediate layers had almost regained the morphological appearance of resting cells with filigrane cytoplasmic processes extended in all directions. The integrin adhesion molecules LFA‐1 (α and β chains) and VLA‐4 were expressed on most microglial cells with activated morphology, as verified by co‐localization with double immunofluorescence labeling for LFA‐1 or VLA‐4 and Griffonia simplicifolia isolectin B4 (GFS‐B4). In contrast, only low levels of integrin adhesion molecule expression were also found on reactive astrocytes along slice surfaces. However, LFA‐1 or VLA‐4 were never found on ramified microglial cells, and double immunofluorescence labeling of LFA‐1 or VLA‐4 with ramified GFS‐B4+ microglia never occurred. We conclude that a) originally resting microglial cells activated in an early phase of in vitro culture but regain a resting status after at least 6 div; and b) integrin adhesion molecules LFA1 and VLA‐4 are potential markers of microglial activation, as they were found on activated but never on resting microglial cells. This enables further investigations on immunological and electrophysiological features of resting and activated microglial cells under in vitro conditions. © 1996 Wiley‐Liss, Inc.

Behavior of in vitro cultured ameboid microglial cells migrating on Müller cell end‐feet in the quail embryo retina

“…5), after microglial cells have carried out MCEF phagocytosis and their cytoplasm is filled with phagosomes. Expression of b1 integrin subunit in activated microglia was previously shown in situ (Hailer et al, 1997;Kloss et al, 1999) and in vitro (Hailer et al, 1996;Kloss et al, 2001;Campbell, 2002, 2003;Nasu-Tada et al, 2005;Zuckerman et al, 1998). b1 integrins are known to be involved in functions of cell adhesion on extracellular matrix (Danen and Sonnenberg, 2003;Elangbam et al, 1997), and the close relationship between its expression in microglial cells and adhesion of these cells to the substrate has been documented (Kloss et al, 1999(Kloss et al, , 2001Campbell, 2002, 2003).…”

Comparison of neuroprotective effects of extract and fractions fromAgarum clathratumagainst experimentally induced transient cerebral ischemic damage

“…Morphological and functional changes in microglia are involved in response to various neural environments (Hailer et al, 1996;Schwartz et al, 2006). Microglia activation is significantly increased in the infarct region induced by focal ischemia and in the CA1 region following transient cerebral ischemia (Mabuchi et al, 2000;Stoll et al, 1998;Sugawara et al, 2002).…”

Cytotoxic CD8+T Cell–Neuron Interactions: Perforin-Dependent Electrical Silencing Precedes But Is Not Causally Linked to Neuronal Cell Death

“…8 E, F ). To study the outcome of OT-I T-cell interactions with antigen-loaded CNS cells including neurons, we used chronic organotypic hippocampal slices (Hailer et al, 1996), which allow incubation of OT-I T cells over a period of 24 h. OT-I T cells were applied to the slice, antigen was added 12 h later, and cocultures were stained with PI and fixed after 24 h. After resectioning, quantitative analysis of cell death was performed by visual identification of PI-positive hippocampal neurons. In organotypic hippocampal slices, antigendependent interactions of OT-I cell with hippocampal neurons resulted in a marked increase in the density of PI-positive neurons (Fig.…”