Research ReportImmunotoxic depletion of microglia in mouse hippocampal slice cultures enhances ischemia-like neurodegeneration
Introduction
Microglial cells, now known to contribute about 12% of the cells in the brain, were first described by del Río-Hortega (1932). Acting as “biosensors” for homeostatic regulation in normal and pathological conditions, microglial cells play a key role in the central nervous reactions to injury, including immune surveillance, extracellular fluid cleansing and cellular debris removal (Dheen et al., 2007, Schloendorn et al., 2007). Resting microglial cells in the adult brain have fine ramified processes, but transform into a non-phagocytic activated phenotype, or a phagocytic activated phenotype when activated (Streit et al., 1999). The normal thin processes then transform into shorter coarse processes (Jensen et al., 1997, Streit et al., 1999), in parallel with the upregulation of normal markers like CD11b (or Mac1) (Perry et al., 1985), MHC class I and II molecules (Kato et al., 1995) and secretion of cytokines like interleukin-1beta (IL-1β) (Buttini et al., 1994), transforming growth factor beta-1 (TGF-β1) (Lehrmann et al., 1998) and tumor necrosis factors-alpha (TNF-α) (Buttini et al., 1996). The reactions to injury also include a drastic increase in the number of microglial cells at the lesion site, due to migration and local proliferation (Neumann et al., 2006).
In relation to microglial activation, it is still not clear to what extent or under which conditions activated forms of microglia exert positive or negative effects on neuronal survival (Li et al., 2007). Following activation microglial cells have thus been reported both to increase neuronal cell death through release of neurotoxic substances like glutamate, toxic cytokines and nitric oxide among others (Chao et al., 1992, Piani et al., 1992, Viviani et al., 1998), and to secrete trophic factors like neurotrophins (Elkabes et al., 1996) and transforming growth factor-β (TGF-β) (Lehrmann et al., 1998).
Experimental depletion of microglial cells before the application of a standardized neurodegenerative insult might help elucidate the role of microglial cells. For this purpose, microglial cells were successfully targeted and eliminated in mouse hippocampal slice cultures by exposure to the ribosome inactivating protein saporin coupled to an antibody towards the microglialy expressed Mac1 receptor (Mac1–sap). When normal and microglia-depleted hippocampal slice cultures thereafter were subjected to 30 min of oxygen–glucose deprivation (OGD), the microglia-depleted cultures displayed an enhanced neuronal degeneration.
Section snippets
Microglia depletion by Mac1–sap treatment
Microglial cells are well represented and display an in vivo-like appearance and distribution in mouse hippocampal slice cultures (Fig. 1A). In order to define parameters for Mac1–sap induced microglia depletion, mouse hippocampal slice cultures grown for 7 days, were exposed to 1.3 nM Mac1–sap, added to the culture medium for 3 or 7 days, thereafter the cultures were immunostained for the microglial marker Mac1 or stained histochemically for microglial tomato lectin (TL) binding. Cultures
Discussion
By this study we have established an easy and efficient way to deplete microglial cells from mouse hippocampal slice cultures, and shown that such microglial deprivation enhanced OGD-induced, CA1 pyramidal cell degeneration.
Mouse hippocampal slice cultures
Seven-day-old C57BL/6J mice were killed by instant decapitation, and the two hippocampi were isolated from the brain and cut in transverse slices at 350 μm by a McIlwain tissue chopper. The hippocampal slices were transferred to chilled Gey's balanced salt solution (Gibco Life Technologies, Paisley, UK) with d-glucose (6.5 mg/ml), trimmed for excess extrahippocampal and meningeal tissues and placed randomly on semiporous (0.4 μm pore size) insert membranes (30 mm in diameter) (Millipore Corp.,
Acknowledgments
Randi Godskesen, Dorte Bramsen and Karen Rich are gratefully acknowledged for their technical assistance. This work was supported by grants from the Danish Research Agency to Prof. Jens Zimmer, including an Internationalization Stipend to Maria Montero, obtained through the Danish Stem Cell Research Doctoral School (DASCDOC).
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